Dock-and-Lock (DNL) Constructs for Human Immunodeficiency Virus (HIV) Therapy

ABSTRACT

The present invention concerns methods and compositions for treatment of HIV infection in a subject, utilizing a DNL complex comprising at least one anti-HIV therapeutic agent, attached to an antibody, antibody fragment or PEG. In a preferred embodiment, the antibody or fragment binds to an antigen selected from gp120, gp41, CD4 and CCR5. In a more preferred embodiment the antibody is P4/D10 or 2G12, although other anti-HIV antibodies are known and may be utilized. In a most preferred embodiment, the anti-HIV therapeutic agent is a fusion inhibitor, such as T20, T61, T651, T1249, T2635, CP32M or T-1444, although other anti-HIV therapeutic agents are known and may be utilized. The DNL complex may be administered alone or may be co-administered with one or more additional anti-HIV therapeutic agents.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/288,202, filed Nov. 3, 2011, which claimed the benefit under 35C.F.R. §119(e) to provisional application Ser. Nos. 61/409,740, filedNov. 3, 2010, and 61/487,956, filed May 19, 2011. This application is acontinuation-in-part of U.S. patent application Ser. No. 11/745,692 (nowU.S. Pat. No. 78,333,971), filed May 8, 2007, which claimed the benefitunder 35 C.F.R. §119(e) to provisional application Ser. No. 60/800,342,filed May 15, 2006. This application is a continuation-in-part of U.S.patent application Ser. Nos. 13/036,820, filed Feb. 28, 2011; 13/021,302(now U.S. Pat. No. 8,246,960), filed Feb. 4, 2011, (which was adivisional of U.S. Pat. No. 7,906,121, which was a divisional of U.S.Pat. No. 7,534,866); 12/968,936, filed Dec. 15, 2010, (which was adivisional of U.S. Pat. No. 7,871,622; which was a divisional of U.S.Pat. No. 7,521,056); 12/964,021, filed Dec. 9, 2010; 12/949,536 (nowU.S. Pat. No. 8,211,440), filed Nov. 18, 2010 (which was a divisionalU.S. Pat. No. 7,858,070, which was a divisional U.S. Pat. No.7,527,787); 12/754,740, filed Apr. 6, 2010; and 12/468,589 (now U.S.Pat. No. 8,163,291), filed May 19, 2009, (which was a divisional of U.S.Pat. No. 7,550,143). Those applications claimed the benefit under 35C.F.R. §119(e) to provisional application Ser. Nos. 61/414,592, filedNov. 17, 2010; 61/302,682, filed Feb. 9, 2010; 61/267,877, filed Dec. 9,2009; 61/168,290, filed Apr. 10, 2009; 60/864,530, filed Nov. 6, 2006;60/782,332, filed Mar. 14, 2006; 60/751,196, filed Dec. 16, 2005;60/728,292, filed Oct. 19, 2005; and 60/668,603, filed Apr. 6, 2005. Thetext of each priority application is incorporated herein by reference inits entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 11, 2011, isnamed IBC129US.txt and is 49,086 bytes in size.

BACKGROUND

1. Field of the Invention

The present invention concerns methods and compositions for treatinghuman immunodeficiency virus (HIV) in infected subjects. Preferably, themethods and compositions utilize complexes made by the dock-and-lock(DNL) technique. In particular embodiments, the DNL complexes compriseantibodies or antibody fragments, which include those against HIVenvelope antigens, for example anti-gp120 or anti-gp41 antibodies suchas P4/D10, 2G12, 2F5 or 4E10, and other antibodies of interest, such asepratuzumab (anti-CD22) and milatuzumab (anti-CD74). In more particularembodiments, the DNL complex may comprise one or more agents, such astherapeutic agents, diagnostic agents, virostatic agents and/orcytotoxic agents, including but not limited to chemotherapeutic agentssuch as doxorubicin. Such agents may be incorporated into the DNLcomplex using the DDD (docking and dimerization domain) and AD(anchoring domain) binding interaction described below, or may bedirectly conjugated to the DNL complex. More preferably, the DNL complexmay comprise one or more agents known to have anti-HIV activity, such asthe T20 (enfuvirtide) HIV fusion inhibitor. Most preferably,incorporation of anti-HIV agents into a DNL complex improves thepharmacokinetic properties of the agent, for example by increasing itsserum half-life, allowing less frequent dosing and/or improved efficacy.In alternative embodiments, the DNL complex may comprise one or morepolyethylene glycol (PEG) moieties to improve pharmacokinetics andreduce immunogenicity. The DNL complexes may be used alone or incombination with one or more known anti-HIV agents.

2. Description of Related Art

Despite encouraging advances in the treatment of human immunodeficiencyvirus-1 (HIV-1) with anti-retroviral therapy (ART), analyses ofperipheral blood and lymph nodes have documented the presence ofpersistent reservoirs of resting T cells which harbor latent provirusthat can activate spontaneously even years after the termination oftherapy (Berger et al., Proc Natl Acad Sci USA 1998, 95:11511-11513;Blankson et al., Annu Rev Med 2002, 53:557-593).

Depending on its binding specificity and effector functions, an antibodycan be useful for preventing the infection of HIV by blocking the viralentry into target cells, evoking complement-mediated virolysis of freevirions (Parren et al., AIDS 1999, 13[Suppl A]:S137-162), and/orinducing Fc receptor-mediated activities (Forthal and Moog, Curr OpinHIV AIDS 2009, 4: 388-393), which include antibody-dependent cellularcytotoxicity (ADCC) to kill infected cells, inhibition andneutralization of HIV on antigen presenting cells, andantibody-dependent cell-mediated virus inhibition (ADCVI).). To date,the use of anti-viral antibodies for immunotherapy of patients infectedwith HIV has not fulfilled its initial promise (Hinkula et al., J AcquirImmune Defic Syndr 1994, 7:940-951; Trkola et al., Nat Med 2005,11:615-622).

Attempts have been made to use various viral or cellular components astargets for antibody delivery of therapeutic agents to HIV-infectedcells (Davey et al., J Infect Dis 1994, 170:1180-1188; Pincus et al., JImmunol 2003, 170:2236-2241; Ramachandran et al., J Infect Dis 1994,170:1009-1013; Saavedra-Lozano et al., Proc Natl Acad Sci USA 2004,101:2494-2499). Similar immunotoxins have proved promising in cancerpatients (Wu and Senter, Nat Biotechnol 2005, 23:1137-1146). However, aneed exists for more effective methods and compositions for treatment ofHIV-infected cells.

SUMMARY OF THE INVENTION

The present invention fulfills an unresolved need in the art byproviding methods and compositions for inhibiting, suppressing,detecting, identifying, localizing and/or eliminating HIV and/orHIV-infected cells. In certain embodiments, the compositions and methodsmay utilize DNL complexes comprising antibodies, antibody fragments orother targeting molecules that bind to HIV antigens. HIV-bindingmolecules may include, but are not limited to, affibodies, monoclonalantibodies, humanized antibodies, chimeric antibodies, human antibodies,antibody fragments and/or antibody analogs. Any antibody or fragmentthereof known in the art that targets HIV or an antigen-presenting cellmay be incorporated into the subject DNL complexes, including but notlimited to P4/D10, 2G12, 2F5, 4E10, and hLL1

In certain embodiments, the HIV targeting molecules may be conjugated toone or more therapeutic and/or diagnostic agents. Such agents mayinclude, but are not limited to, a drug, prodrug, virostatic agent,toxin, enzyme, oligonucleotide, radioisotope, radionuclide,immunomodulator, cytokine, label, fluorescent label, luminescent label,paramagnetic label, MRI label, micelle, liposome, nanoparticle, orcombination thereof. In alternative embodiments, the HIV targetingmolecules may be attached to therapeutic agents via the DNL technologydescribed below.

The DNL complexes may be administered to patients with a known orsuspected HIV infection. Administration may be by any route known in theart, such as orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal, intraarterial, intrathecal or intravenous injection.Alternatively, administration may be oral, nasal, buccal, inhalational,rectal, vaginal or topical. Such administration may destroy HIV incirculation, may block or prevent infection of cells by HIV, may reduceor eliminate HIV-infected cells in the patient, and/or may reduce oreliminate residual foci of HIV-infected cells in patients treatedpreviously and/or simultaneously with other known anti-retroviraltherapies.

The skilled artisan will realize that the subject DNL complexes may beadministered either alone or in combination with other known therapeutictreatments for HIV infection, such as azidothymidine, othernucleoside/nucleotide reverse transcriptase inhibitors, non-nucleosidereverse transcriptase inhibitors, HIV protease inhibitors and/or fusioninhibitors. In certain embodiments, the conjugated HIV targetingmolecules may be used in combination with HAART (highly activeanti-retroviral therapy). Many anti-HIV therapeutic agents are known inthe art and any such known agent may be used, including but not limitedto abacavir, amdoxovir, apricitabine, atazanavir, bevirimat, calanolideA, CCR5, CD4, ceragenin, cobicistat, cyanovirin-N, darunavir,diarylpyrimidines, didanosine, dolutegravir, efavirenz, elvitegravir,elvucitabine, emtricitabine, epigallotachen gallate, festinavir,fosamprenavir, foscarnet, griffithsin, globoidnan A, hydroxycarbamide,indinavir, KP-146, lamivudine, lefinavir, lersivirine, lopinavir,miltefosine, MK-2048, nelfinavir, nevirapine, racivir, raltegravir,ritonavir, saquinavir, selicicib, stafudine, stampidine, stavudine, Tatantagonists, tenofovir, tipranavir, trichosanthin, TRIM5alpha, vivecon,zalcitabine, zidovudine or zidovudine, either alone or in anycombination.

The subject DNL complexes may comprise an antibody or antibody fragmentof interest attached to multiple copies of a toxin or a peptide-basedfusion inhibitor. The toxins may be of a microbial, plant, or animalorigin, including and not limited to ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin,Pseudomonas endotoxin, ranpirnase (Rap) or Rap (N69Q). Peptide-basedfusion inhibitors (Naider and Anglister, Curr Opin Struct Biol 2009, 19:473-482) include but are not limited to those targeting the C-terminalhelical region of gp41, for example, T-20, T1249, C34, DP, andsifuvirtide, or those targeting the N-terminal helical region of gp41,for example, IZN17, N17, N38, N42, N36F10, and T21. More preferably,such DNL complexes display anti-HIV activity at nanomolar or lowerconcentrations.

Yet another embodiment relates to DNL complexes for delivery oftherapeutic nucleic acid species, such as artificial genes or siRNA. Insuch embodiments, the DNL complex may comprise an anti-HIV antibody orfragment thereof attached to one or more copies of a nucleic acidcarrier, such as a dendrimer, a protamine, a histone,histidine-containing reducible polycation, cationic comb-type copolymer,chitosan-thiamine pyrophosphate, polyethyleneimine or polylysine. Manyexamples of nucleic acid binding polymers are known in the art, such asPAMAM, polylysine, polypropyleneimine, polyethyleneimine,polyethyleneglycol or carbosilane. Generally, the carrier molecule ispolycationic and binds to nucleic acids by electrostatic interaction. Asdiscussed below, many examples of siRNA or other therapeutic nucleicacids are known in the art and any such known species may be deliveredto a target cell, tissue, organ or pathogen using the DNL complexesdescribed herein.

The subject (DNL) complexes comprise at least two copies of adimerization and docking domain (DDD) moiety and at least one copy of ananchoring domain (AD) moiety. Preferably, the DDD moiety is from a humanprotein kinase A regulatory subunit protein (RIα, RIβ, RIIα, RIM) whilethe AD moiety is from an AKAP (A-kinase anchoring protein). The DDDmoieties spontaneously form dimers which then bind to the AD moiety toform the DNL complex. The DNL complexes may comprise fusion proteinsincorporating the AD and DDD moieties, although alternatively the ADand/or DDD moieties may be covalently attached to effector moieties byother methods, such as chemical coupling. Effectors incorporated intothe DNL complex may include, but are not limited to, proteins, peptides,antibodies, antibody fragments, immunomodulators, cytokines,interleukins, interferons, binding proteins, peptide ligands, carrierproteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule knownto produce physiological effects. The subject DNL complexes may becomprised of dimers, trimers, tetramers, pentamers, hexamers or othermultimers. The skilled artisan will realize that the DNL technologyallows the efficient and reproducible formation of multimeric complexescomprising virtually any combination of effector subunits.

Also described herein are isolated nucleic acids encoding a fusionprotein or other DNL subunit, as described herein. Other embodimentsconcern expression vectors and/or host cells comprising the encodingnucleic acid sequences. In certain preferred embodiments, the host cellmay be an Sp2/0 cell line transformed with a mutant Bcl-2 gene, forexample with a triple mutant Bcl-2 gene (T69E, S70E, S87E), that hasbeen adapted to cell transformation and growth in serum free medium.(See, e.g., U.S. Pat. Nos. 7,531,327; 7,537,930; and 7,608,425, theExamples section of each of which is incorporated herein by reference.)The host cell transfected with expression vector(s) encoding a DNLcomplex, or a subunit of a DNL complex, may be cultured by standardtechniques for production of the encoded protein or complex.Advantageously, the host cell is adapted for growth and proteinproduction under serum-free conditions.

The skilled artisan will realize that the DNL complexes and uses thereofdisclosed above are exemplary only and that many other different typesof DNL complexes, for either therapeutic or diagnostic use, are includedwithin the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of particularembodiments of the invention. The embodiments may be better understoodby reference to one or more of these drawings in combination with thedetailed description presented herein.

FIG. 1A. Neutralization of HIV infection in vitro. The neutralizingcapacities of the immunoglobulins were tested by incubating differentconcentrations of the immunoglobulins with the HIV-1_(IIIB) laboratorystrain and then assaying the viral infection of HIV-susceptible JurkatT-cells. Both 10 μg/ml doxorubicin-P4/D10 and unlabelled P4/D10neutralized HIV-1_(IIIB) significantly better than HIV negative sera(p=0.001).

FIG. 1B. Inhibition of intercellular spread of HIV infection in vitro.To test whether the immunoglobulins could limit the intercellular spreadof HIV-1 infection, Jurkat T-cells were mixed in the proportions of0.2%, 1%, 3%, and 5% infected and 99.8%, 99%, 97%, and 95% uninfectedcells. The HIV-1 p24 production after treating 3% Jurkat T-cellsinfected with HIV-1_(IIIB) and 97% uninfected cells with differentconcentrations of immunoglobulins is shown. The results are shown aspercent inhibition of p24 production after 7 days in culture.Doxorubicin-P4/D10 had a significantly better inhibiting effect onproduction of HIV-1 p24 compared to unlabelled P4/D10, control antibodydoxorubicin-LL1, free doxorubicin and HIV-negative serum at aconcentration of 0.5 or 0.05 μg/ml (p=0.002).

FIG. 2. Protection against HIV-1/MuLV infection in vivo. Mice(6-12/group) were challenged i.p. with HIV-1/MuLV infected splenocytesand immediately treated with monoclonal antibodies (MAb) or freedoxorubicin. Unconjugated P4/D10 MAb was titrated 100-800 μg per mouse,free doxorubicin 100-400 μg and irrelevant doxorubicin-hRS7 100-200 μg.All other treatments were given at 100 μg per mouse. Ten days afterchallenge, peritoneal cells were collected and mixed with HIVsusceptible Jurkat T-cells. HIV p24 production in these cell cultureswas measured every 3-4 days for 18 days. Percent of mice with a p24positive cell culture after treatment with 100 μg of MAb or freedoxorubicin is shown. Only cells from mice treated with 100 μgdoxorubicin-P4/D10 contained no infectious HIV, which was significantlydifferent (p=0.0001) from all other groups.

FIG. 3. Analysis of Hex-hA20 binding. (A) Competition ELISA showingHex-hA20 has a higher avidity than veltuzumab for binding to WR2.Hex-hA20 (∘) or veltuzumab (▪) were incubated at varying concentrationsin the presence of WR2 for competition of binding with immobilizedveltuzumab. The percentage of inhibition was plotted versus mAbconcentration, and EC₅₀ values were generated with PRISM® software. (B)Binding to Daudi cells as determined by flow cytometry usingPE-conjugated anti-human Fab (PE-anti-Fab) or PE-conjugated anti-humanFc (PE-anti-Fc). All incubations and washes were performed at 4° C.Daudi cells were suspended at 1×10⁶ cells/mL in 1% BSA-PBS and incubatedwith Hex-hA20, veltuzumab, or labetuzumab for 1 h. The cells were washedwith 1% BSA-PBS, incubated with a 1:200 dilution of PE-anti-Fab orPE-anti-Fc for 30 min, washed once more, and analyzed on a GUAVA® PCA.(C) Scatchard analysis using radio-iodinated Hex-hA20 (▪), veltuzumab (), or rituximab (∘) and Raji cells. (D) Dissociation from Daudi or Rajicells. Hex-hA20 (), veltuzumab (▪), and rituximab ( ) were labeled withPE using a ZENON® R-phycoerythrin Human IgG Labeling kit (InvitrogenCorp.). Cells were suspended in CM (phenol red-free RPMI 1640supplemented with 10% FBS at 1×10⁶ cells/mL), and 5×10⁵ cells wereincubated with each PE-labeled antibody at 65 nmol/L for 30 min at roomtemperature. The cells were washed twice with CM to remove unboundantibody, resuspended in 1.5 mL of CM in the presence of 1 μmol/LC_(H1)-DDD2-Fab-hA20 at 37° C., and analyzed for cell-bound PE-labeledantibody at several time points on a GUAVA® PCA. The dissociationhalf-life was determined by nonlinear regression using PRISM® software.

FIG. 4. Inhibition of cell proliferation. (A) In vitro antiproliferationdetermined by the 4-d MTS assay for Raji, Ramos, or Daudi. Cells weretreated with Hex-hA20 (∘), veltuzumab ( ), or veltuzumab plus goatanti-human Fc (▪). Daudi cells were also treated with Hex-hA20 plus goatanti-human Fc ( ). Briefly, cells were placed in 96-well plates at 5,000cells per well in complete RPMI 1640. Five-fold serial dilutions ofHex-hA20, veltuzumab, or veltuzumab cross-linked with goat-anti human Fcwere added to triplicate wells at final concentrations ranging from2×10⁻⁸ to 6.4×10⁻¹² mol/L. The plates were incubated for 4 d, afterwhich 20 μL of CELLTITER 96® Aqueous One Solution Reagent (PromegaCorp.) was added, and the incubation was continued for an additional 4 hbefore reading the plates at 490 nm. (B) In vitro antiproliferationdetermined by the viable cell counting assay for Ramos (left) or Raji(right). Cells were seeded in T-flasks at 1×10⁵ cells/mL and treatedwith veltuzumab, Tri-hA20, Tetra-hA20, or Hex-hA20 at the indicatedconcentrations. Viable cell densities (VCD) were determined daily over 5d by flow cytometry. On day 3, cultures were split 1:2 to maintainlogarithmic growth. Cells were plotted as viable cells per millilitermeasured on days 3, 4, and 5 at the indicated concentrations.

FIG. 5 (A) Apoptosis measured by GUAVA® Nexin (left) showing percentageof early apoptotic cells (Annexin V-PE positive/7-AAD negative) inducedin Raji after 24-h incubation with veltuzumab, Tri-hA20, Tetra-hA20, orHex-hA20 at 0.5 nmol/L (black columns) or 5 nmol/L (gray columns).Apoptosis measured by GUAVA® MultiCaspase (right) for which Raji cellswere cultured in the presence of Hex-hA20 (5 nmol/L), veltuzumab (5nmol/L), or anti-IgM (5 μg/mL) and analyzed at 3, 7, 16, and 24 h byflow cytometry after staining with SR-VAD-FMK. Cells were plated at2×10⁵ cells/mL in fresh media and incubated at 37° C. with each testarticle at the indicated concentrations for up to 24 h, and duplicatewells were processed for GUAVA® analysis. (B) CDC (left) was measured inDaudi cells for Hex-hA20 (∘), epratuzumab □( ), veltuzumab (▪), orC_(H3)-AD2-IgG-hA20 () in the presence of human complement. Thepercentage complement control (number of viable cells in the test samplecompared with cells treated with complement only) was plotted versus thelog of the nanomolar concentration. ADCC (right) was measured forHex-hA20, veltuzumab, epratuzumab, or labetuzumab at 5 μg/mL using Daudias the target cells and freshly isolated peripheral blood mononuclearcells from two donors as the effector cells. A 100% lysis reference wasgenerated by the addition of detergent to wells containing target cellsonly. The bar graphs show percentage of lysis obtained for each of thetwo donors.

FIG. 6. Efficacy of Hex-hA20 in human lymphoma xenograft models. (A)Daudi cells (1.5×10⁷) were injected i.v. into SCID mice on day 0. Ondays 1 and 8, groups of mice (n=9-10) were given either Hex-hA20 at twodifferent doses (30 or 6 μg) or equimolar amounts of veltuzumab (12.4 or2.4 μg). (B) SCID mice were depleted of NK cells and neutrophils beforethe administration of Raji cells with antimouse Gr-1 ascites and TMβ-1mAb specific for mouse IL-2 receptor, as described in Materials andMethods. On day 0, Raji cells (1×10⁶) were injected i.v. into bothdepleted and nondepleted mice. Hex-hA20 (465 μg) or veltuzumab (200 μg)was given i.v. on days 3, 5, 7, and 11, whereas the control groupreceived saline.

FIG. 7. Schematic diagram of IgG-(T20)₄ DNL complex. (A) Amino acidsequences of AD2 (SEQ ID NO:4) and DDD2 (SEQ ID NO:2) moieties. (B)Amino acid sequence of DDD2-linker-poly-histidine-T20 moiety (SEQ IDNO:99). (C) Structures of IgG-AD2 and DDD2-T20 subunits and DNL complex.

FIG. 8. Amino acid sequences of (A) V_(K) chain (SEQ ID NO:100) and (B)V_(H) chain (SEQ ID NO:101) of P4/D10 antibody. The CDR sequences areunderlined.

FIG. 9A. Nucleotide and amino acid sequences of chimeric P4/D10(cP4/D10) antibody light and heavy chain variable regions. The aminoacid variable region sequences of the chimeric antibody are identical tothose of the murine P4/D10 antibody. (A) DNA sequence of chimeric V_(K)chain (SEQ ID NO:102). (B) Amino acid sequence of chimeric V_(K) chain(SEQ ID NO:103). (C) DNA sequence of chimeric V_(H) chain (SEQ IDNO:104). (D) Amino acid sequence of chimeric V_(H) chain (SEQ IDNO:105).

FIG. 10. Comparison of binding of cP4/D10 and P4/D10. (A) ELISA assay ofbinding to the HIV envelope protein gp160 coated on microtiter plates.(B) ELISA assay of binding to the V-3 peptide of gp120.

FIG. 11. Inhibition of HIV-1₆₉₂₀ replication in PBMCs by h734-(T20)₄,DDD2-T20 and T20 (FUZEON®) as determined by p24 antigen ELISA at day 9.(A) The concentrations of the test articles in μg/mL were used for theX-axis. (B) The superior potency of h734-(T20)₄ compared to DDD2-T20 andT20 was revealed when the molar concentrations of the test articles wereused for the X-axis.

FIG. 12. Comparing the potency of P4/D10, cP4/D10, h734-(T20)₄, andhLL2-(T20)₄ for neutralizing HIV. (A) Jurkat T cells exposed toHIV-1_(IIIB) were dosed at 50 TCID₅₀. (B) Jurkat T cells exposed toHIV-1_(IIIB) were dosed at 100 TCID₅₀. (C) PBMCs exposed to HIV-1₆₇₉₄were dosed at 50 TCID₅₀. (D) PBMCs exposed to HIV-1₆₇₉₄ were dosed at100 TCID₅₀.

FIG. 13. Neutralization of HIV-1 in PBMCs following activation of latentvirus by SAHA over a period of 30 days. (A) HIV-1 was monitored by p24antigen capture. (B) HIV-1 was monitored by number of HIV-positivecultures. (C) The virus-positive cultures on Day 30 in cells treatedwith each agent are shown in as a percent of the medium-treated control.

FIG. 14. Serum stability of hLL2-(T20)₄. Concentrations of intacthLL2-(T20)₄ and all hLL2-containing species in serum samples collectedfrom mice at 30-min, 6-h, 24-h, and 72-h, post-injection of hLL2-(T20)₄,compared with concentrations of hLL2 in serum samples collected frommice at the same time points post-injection of hLL2.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety.

DEFINITIONS

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

As used herein, “about” means within plus or minus ten percent of anumber. For example, “about 100” would mean any number between 90 and110.

An “antibody”, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion or analog of an immunoglobulin molecule, like an antibodyfragment.

An “antibody fragment” is a portion of an antibody such as F(ab)₂,F(ab′)₂, Fab, Fv, sFv, and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. The term “antibody fragment” also includes anysynthetic or genetically engineered protein that acts like an antibodyby binding to a specific antigen to form a complex. For example,antibody fragments include isolated fragments consisting of the variableregions, such as the “Fv” fragments consisting of the variable regionsof the heavy and light chains, recombinant single chain polypeptidemolecules in which light and heavy variable regions are connected by apeptide linker (“scFv proteins”), and minimal recognition (CDR) unitsconsisting of the amino acid residues that mimic the hypervariableregion.

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includeantibodies, antibody fragments, drugs, virostatic agents, toxins,enzymes, nucleases, hormones, immunomodulators, antisenseoligonucleotides, small interfering RNA (siRNA), chelators, boroncompounds, photoactive agents, dyes, and radioisotopes. Other exemplarytherapeutic agents and methods of use are disclosed in U.S. PatentApplication Publication Nos. 20050002945, 20040018557, 20030148409 and20050014207, each incorporated herein by reference.

A “neutralizing antibody” or “neutralizing antibody fragment” is usedherein to refer to an antibody or fragment that reacts with aninfectious agent (such as a virus) and inhibits its infectivity.

A “diagnostic agent” is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes (such as with the biotin-streptavidincomplex), contrast agents, fluorescent compounds or molecules, andenhancing agents (e.g., paramagnetic ions) for magnetic resonanceimaging (MRI).

An “immunoconjugate” is a conjugate of a binding molecule (e.g., anantibody component) with an atom, molecule, or a higher-orderedstructure (e.g., with a carrier, a therapeutic agent, or a diagnosticagent).

A “naked antibody” is an antibody that is not conjugated to any otheragent.

A “carrier” is an atom, molecule, or higher-ordered structure that iscapable of associating with a therapeutic or diagnostic agent tofacilitate delivery of such agent to a targeted cell. Carriers mayinclude lipids (e.g., amphiphilic lipids that are capable of forminghigher-ordered structures), polysaccharides (such as dextran), proteins,peptides, peptide analogs, peptide derivatives or other higher-orderedstructures, such as micelles, liposomes, or nanoparticles. In certainembodiments, a carrier may be designed to be resistant to proteolytic orother enzymatic degradation, for example by substituting D-amino acidsfor naturally occurring L-amino acids in a protein or peptide.

As used herein, the term “antibody fusion protein” refers to arecombinantly produced antigen-binding molecule in which two or more ofthe same or different scFv or antibody fragments with the same ordifferent specificities are linked. Valency of the fusion proteinindicates how many binding arms or sites the fusion protein has to asingle antigen or epitope; i.e., monovalent, bivalent, trivalent ormultivalent. The multivalency of the antibody fusion protein means thatit can take advantage of multiple interactions in binding to an antigen,thus increasing the avidity of binding to the antigen. Specificityindicates how many antigens or epitopes an antibody fusion protein isable to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Monospecific, multivalent fusionproteins have more than one binding site for an epitope but only bindsto one such epitope, for example a diabody with two binding sitereactive with the same antigen. The fusion protein may comprise a singleantibody component, a multivalent or multispecific combination ofdifferent antibody components, or multiple copies of the same antibodycomponent. The fusion protein may additionally comprise an antibody oran antibody fragment and a therapeutic agent. Examples of therapeuticagents suitable for such fusion proteins include immunomodulators(“antibody-immunomodulator fusion protein”) and toxins (“antibody-toxinfusion protein”). One preferred toxin comprises a ribonuclease (RNase),preferably a recombinant RNase.

An antibody or immunoconjugate preparation, or a composition describedherein, is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient mammal. Inparticular, an anti-HIV antibody preparation is physiologicallysignificant if its presence reduces, inhibits or eliminates HIV-infectedcells or reduces, inhibits or eliminates HIV infection of non-infectedcells.

A composition is said to be a “pharmaceutically acceptable carrier” ifits administration can be tolerated by a recipient patient. Sterilephosphate-buffered saline is one example of a pharmaceuticallyacceptable carrier. Other suitable carriers are well known to those inthe art. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed.(Mack Publishing Co. 1995), and Goodman and Gilman's THE PHARMACOLOGICALBASIS OF THERAPEUTICS (Goodman et al., Eds. Macmillan Publishing Co.,New York, 1980 and 2001 editions).

Abbreviations used are:

-   -   ABS, sodium acetate buffer containing 150 mM sodium chloride;    -   ADCC, antibody-dependent cell-mediated cytotoxicity;    -   DNL, Dock-and-Lock;    -   DTT, dithiothreitol;    -   ELISA, enzyme-linked immunosorbent assay;    -   ART, anti-retroviral therapy;    -   HIV, human immunodeficiency virus;    -   MAb or mAb, monoclonal antibody;    -   MuLV, Murine Leukemia Virus;    -   PBMC, peripheral blood mononuclear cells;    -   TCID₅₀, 50% tissue culture infectious dose

The DNL method has been used to prepare a wide variety of multimericconstructs (see, e.g., U.S. Pat. Nos. 7,521,056; 7,527,787; 7,534,866;7,550,143 and 7,666,400, the Examples section of each of which isincorporated herein by reference.) The DNL method is capable of joiningvirtually any effector subunit(s) of interest in a stable complex, withvery high reproducibility and efficiency. Generally, DNL takes advantageof the specific and high-affinity binding interaction between adimerization and docking domain (DDD) sequence derived fromcAMP-dependent protein kinase regulatory subunit and an anchor domain(AD) sequence derived from any of a variety of AKAP proteins. The DDDand AD peptides may be attached to any protein, peptide or othermolecule. Because the DDD sequences spontaneously dimerize and bind tothe AD sequence, the DNL technique allows the formation of complexesbetween any selected molecules that may be attached to DDD or ADsequences. Although the standard DNL complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers.

In some embodiments, the DNL complex may comprise two or moreantibodies, antibody fragments or fusion proteins which bind todifferent epitopes of the same antigen or to two or more differentantigens. The DNL complex may also comprise one or more other effectors,such as proteins, peptides, immunomodulators, cytokines, interleukins,interferons, binding proteins, peptide ligands, carrier proteins,toxins, ribonucleases such as onconase, inhibitory oligonucleotides suchas siRNA, polymers such as PEG, enzymes, therapeutic agents, hormones,cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents or anyother molecule or aggregate.

The DNL method exploits specific protein/protein interactions that occurbetween the regulatory (R) subunits of cAMP-dependent protein kinase(PKA) and the anchoring domain (AD) of A-kinase anchoring proteins(AKAPs) (Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott,Nat. Rev. Mol. Cell. Biol. 2004; 5: 959). PKA, which plays a centralrole in one of the best studied signal transduction pathways triggeredby the binding of the second messenger cAMP to the R subunits, was firstisolated from rabbit skeletal muscle in 1968 (Walsh et al., J. Biol.Chem. 1968; 243:3763). The structure of the holoenzyme consists of twocatalytic subunits held in an inactive form by the R subunits (Taylor,J. Biol. Chem. 1989; 264:8443). Isozymes of PKA are found with two typesof R subunits (RI and RII), and each type has α and isoforms (Scott,Pharmacol. Ther. 1991; 50:123). Thus, the four isoforms of PKAregulatory subunits are RIα, RIβ, RIIα and RIIβ. The R subunits havebeen isolated only as stable dimers and the dimerization domain has beenshown to consist of the first 44 amino-terminal residues (Newlon et al.,Nat. Struct. Biol. 1999; 6:222). Binding of cAMP to the R subunits leadsto the release of active catalytic subunits for a broad spectrum ofserine/threonine kinase activities, which are oriented toward selectedsubstrates through the compartmentalization of PKA via its docking withAKAPs (Scott et al., J. Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunit and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNLcomplex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the “dock-and-lock” approach is asfollows. Entity A is constructed by linking a DDD sequence to aprecursor of A, resulting in a first component hereafter referred to asa. Because the DDD sequence would effect the spontaneous formation of adimer, A would thus be composed of a₂. Entity B is constructed bylinking an AD sequence to a precursor of B, resulting in a secondcomponent hereafter referred to as b. The dimeric motif of DDD containedin a₂ will create a docking site for binding to the AD sequencecontained in b, thus facilitating a ready association of a₂ and b toform a binary, trimeric complex composed of a₁b. This binding event ismade irreversible with a subsequent reaction to covalently secure thetwo entities via disulfide bridges, which occurs very efficiently basedon the principle of effective local concentration because the initialbinding interactions should bring the reactive thiol groups placed ontoboth the DDD and AD into proximity (Chmura et al., Proc. Natl. Acad.Sci. USA. 2001; 98:8480) to ligate site-specifically. Using variouscombinations of linkers, adaptor modules and precursors, a wide varietyof DNL constructs of different stoichiometry may be produced and used,including but not limited to dimeric, trimeric, tetrameric, pentamericand hexameric DNL constructs (see, e.g., U.S. Pat. Nos. 7,550,143;7,521,056; 7,534,866; 7,527,787 and 7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNLconstruct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL constructs, different AD or DDD sequences maybe utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 5) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEA KDDD3C (SEQ ID NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC 

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 8) SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK PKA RIβ (SEQ ID NO: 9)SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEEN RQILA PKA RIIα(SEQ ID NO: 10) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RIIβ(SEQ ID NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Can et al., 2001, J Biol Chem 276:17332-38; Altoet al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker et al.,2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J 400:493-99;Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell24:397-408, the entire text of each of which is incorporated herein byreference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:1 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:1are shown in Table 1. In devising Table 1, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. Even withsuch conservative substitutions, there are over twenty million possiblealternative sequences for the 44 residue peptide(2×3×2×2×2×2×2×2×2×2×2×2×2×2×2×4×2×2×2×2×2×4×2×4). A limited number ofsuch potential alternative DDD moiety sequences are shown in SEQ IDNO:12 to SEQ ID NO:31 below. The skilled artisan will realize that analmost unlimited number of alternative species within the genus of DDDmoieties can be constructed by standard techniques, for example using acommercial peptide synthesizer or well known site-directed mutagenesistechniques. The effect of the amino acid substitutions on AD moietybinding may also be readily determined by standard binding assays, forexample as disclosed in Alto et al. (2003, Proc Natl Acad Sci USA100:4445-50).

TABLE 1 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1). Consensus sequence disclosed as SEQ ID NO: 90. S H I Q I P P G L T E L LQ G Y T V E V L R T K N A S D N A S D K R Q Q P P D L V E F A V E Y F TR L R E A R A N N E D L D S K K D L K L I I I V V VTHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 12)SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 13)SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 14)SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 15)SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 16)SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 17)SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 18)SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 19)SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 20)SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 21)SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFIRLREARA (SEQ ID NO: 22)SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 23)SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 24)SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 25)SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 26)SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA (SEQ ID NO: 27)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA (SEQ ID NO: 28)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 29)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 30)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA (SEQ ID NO: 31)

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:3), with abinding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed asa peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:3 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 2 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:3), similar tothat shown for DDD1 (SEQ ID NO:1) in Table 1 above.

Even with such conservative substitutions, there are over thirty-fivethousand possible alternative sequences for the 17 residue AD1 (SEQ IDNO:3) peptide sequence (2×3×2×4×3×2×2×2×2×2×2×4). A limited number ofsuch potential alternative AD moiety sequences are shown in SEQ ID NO:32to SEQ ID NO:49 below. Again, a very large number of species within thegenus of possible AD moiety sequences could be made, tested and used bythe skilled artisan, based on the data of Alto et al. (2003). It isnoted that FIG. 2 of Alto (2003) shows an even large number of potentialamino acid substitutions that may be made, while retaining bindingactivity to DDD moieties, based on actual binding experiments.

AKAP-IS QIEYLAKQIVDNAIQQA (SEQ ID NO: 3)

TABLE 2 Conservative Amino Acid   Substitutions in AD1 (SEQ ID NO: 3). Consensus sequence   disclosed as SEQ ID NO: 91. Q I E Y L A KQ I V D N A I Q Q A N L D F I R N E Q N N L V T V I S VNIEYLAKQIVDNAIQQA (SEQ ID NO: 32) QLEYLAKQIVDNAIQQA (SEQ ID NO: 33)QVEYLAKQIVDNAIQQA (SEQ ID NO: 34) QIDYLAKQIVDNAIQQA (SEQ ID NO: 35)QIEFLAKQIVDNAIQQA (SEQ ID NO: 36) QIETLAKQIVDNAIQQA (SEQ ID NO: 37)QIESLAKQIVDNAIQQA (SEQ ID NO: 38) QIEYIAKQIVDNAIQQA (SEQ ID NO: 39)QIEYVAKQIVDNAIQQA (SEQ ID NO: 40) QIEYLARQIVDNAIQQA (SEQ ID NO: 41)QIEYLAKNIVDNAIQQA (SEQ ID NO: 42) QIEYLAKQIVENAIQQA (SEQ ID NO: 43)QIEYLAKQIVDQAIQQA (SEQ ID NO: 44) QIEYLAKQIVDNAINQA (SEQ ID NO: 45)QIEYLAKQIVDNAIQNA (SEQ ID NO: 46) QIEYLAKQIVDNAIQQL (SEQ ID NO: 47)QIEYLAKQIVDNAIQQI (SEQ ID NO: 48) QIEYLAKQIVDNAIQQV (SEQ ID NO: 49)

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:50),exhibiting a five order of magnitude higher selectivity for the RHisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-teuuinal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS QIEYVAKQIVDYAIHQA (SEQ ID NO: 50)Alternative AKAP sequences QIEYKAKQIVDHAIHQA (SEQ ID NO: 51)QIEYHAKQIVDHAIHQA (SEQ ID NO: 52) QIEYVAKQIVDHAIHQA (SEQ ID NO: 53)

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs AKAP-KL PLEYQAGLLVQNAIQQAI (SEQ ID NO: 54) AKAP79LLIETASSLVKNAIQLSI (SEQ ID NO: 55) AKAP-Lbc LIEEAASRIVDAVIEQVK(SEQ ID NO: 56) RI-Specific AKAPs AKAPce ALYQFADRFSELVISEAL(SEQ ID NO: 57) RIAD LEQVANQLADQIIKEAT (SEQ ID NO: 58) PV38FEELAWKIAKMIWSDVF (SEQ ID NO: 59) Dual-Specificity AKAPs AKAP7ELVRLSKRLVENAVLKAV (SEQ ID NO: 60) MAP2D TAEEVSARIVQVVTAEAV(SEQ ID NO: 61) DAKAP1 QIKQAAFQLISQVILEAT (SEQ ID NO: 62) DAKAP2LAWKIAKMIVSDVMQQ (SEQ ID NO: 63)

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:64-66. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:64), RIAD (SEQ IDNO:65) and PV-38 (SEQ ID NO:66). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 DLIEEAASRIVDAVIEQVKAAGAY (SEQ ID NO: 64) RIAD LEQYANQLADQIIKEATE(SEQ ID NO: 65) PV-38 FEELAWKIAKMIWSDVFQQC (SEQ ID NO: 66)

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 3 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 3 AKAP Peptide sequences Peptide Sequence AKAPIS QIEYLAKQIVDNAIQQA(SEQ ID NO: 3) AKAPIS-P QIEYLAKQIPDNAIQQA (SEQ ID NO: 67) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:3). The residues are the same as observed byAlto et al. (2003), with the addition of the C-terminal alanine residue.(See FIG. 4 of Hundsrucker et al. (2006), incorporated herein byreference.) The sequences of peptide antagonists with particularly highaffinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS QIEYLAKQIVDNAIQQA (SEQ ID NO: 3)

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:1. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:1) sequence, based on the data of Carr et al. (2001) is shownin Table 4. Even with this reduced set of substituted sequences, thereare over 65,000 possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 1 andTable 2.

TABLE 4 Conservative Amino Acid Substitutionsin DDD1 (SEQ ID NO: 1). Consensus sequence disclosed as SEQ ID NO: 92. SH I Q I P P G L T E L L Q G Y T V E V L R T N S I L A Q Q P P D L V E FA V E Y F T R L R E A R A N I D S K K L L L I I A V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+-0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gin, asn, lys; Asn (N) his, asp, lys, arg, gin; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Antibodies

Various embodiments may concern antibodies and/or antibody fragmentsthat bind to one or more antigens or epitopes of HIV. In preferredembodiments, the antigen or epitope is one that is exposed on thesurface of HIV-infected cells, such as the HIV envelope protein.Alternatively, the antigen or epitope may be one that is displayed onthe surface of an HIV-infected cell. Techniques for preparing and usingvarious antibody-based constructs and fragments are well known in theart. Means for preparing and characterizing antibodies are also wellknown in the art (See, e.g., Harlowe and Lane, 1988, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory). Antibodies of use mayalso be commercially obtained from a wide variety of known sources. Forexample, a variety of antibody secreting hybridoma lines are availablefrom the American Type Culture Collection (ATCC, Manassas, Va.).

Monoclonal Antibodies

While preferred embodiments may concern the use of the P4/D10 antibody,other anti-HIV antibodies may be obtained, prepared and/or used. Avariety of antibodies against HIV have been reported and in certainembodiments any such known anti-HIV antibody may be utilized. Forexample, 4E10 (Rosa et al., Immunity 2:163-73, 2005); 2F5 (Bryson etal., Protein and Peptide Letters, 8:413-18, 2001); 3D6 (Ruker et al.,Ann. NY Acad. Sci. 646:212-19, 1991); C37 (Cao et al., DNA and CellBiology, 12:836-41, 2004); 1ACY, 1F58, 1GGGC (Berry et al., Proteins,45:281-82, 2001); 2G12 (Armbruster et al., J. Antimicrob. Chemother.54:915-20, 2004), each incorporated herein by reference. In alternativeembodiments, monoclonal antibodies may be readily prepared through useof well-known techniques, such as those exemplified in U.S. Pat. No.4,196,265. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition. Cells from rodents such asmice and rats are preferred. Mice are more preferred, with the BALB/cmouse being most preferred as this is most routinely used and generallygives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Often, a panel of animals will have been immunized and thespleen of the animal with the highest antibody titer will be removed andthe spleen lymphocytes obtained by homogenizing the spleen with asyringe. Typically, a spleen from an immunized mouse containsapproximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B-lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art. For example, where the immunized animal is a mouse,one may use P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one mayuse R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6 are all useful in connection with cellfusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus, and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, have been described. The use ofelectrically induced fusion methods is also appropriate.

Fusion procedures usually produce viable hybrids at low frequencies,around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryagents are aminopterin, methotrexate, and azaserine. Aminopterin andmethotrexate block de novo synthesis of both purines and pyrimidines,whereas azaserine blocks only purine synthesis. Where aminopterin ormethotrexate is used, the media is supplemented with hypoxanthine andthymidine as a source of nucleotides (HAT medium). Where azaserine isused, the media is supplemented with hypoxanthine.

A preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two wk. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three wk) for the desired reactivity. The assay should be sensitive,simple and rapid, such as radioimmunoassays, enzyme immunoassays,cytotoxicity assays, plaque assays, dot immunobinding assays, and thelike.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide MAbs in high concentration. The individualcell lines also could be cultured in vitro, where the MAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. MAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation, and variouschromatographic methods such as HPLC or affinity chromatography.

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Such antibody fragments may be obtained by pepsin orpapain digestion of whole antibodies by conventional methods. Forexample, antibody fragments may be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment may be further cleaved using a thiol reducing agent and,optionally, a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment. Exemplary methods forproducing antibody fragments are disclosed in U.S. Pat. No. 4,036,945;U.S. Pat. No. 4,331,647; Nisonoff et al., 1960, Arch. Biochem. Biophys.,89:230; Porter, 1959, Biochem. J., 73:119; Edelman et al., 1967, METHODSIN ENZYMOLOGY, page 422 (Academic Press), and Coligan et al. (eds.),1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(I), chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotidelinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingsFvs are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780). Single domain antibodies may be obtained,for example, from camels, alpacas or llamas by standard immunizationtechniques. (See, e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yauet al., J Immunol Methods 281:161-75, 2003; Maass et al., J ImmunolMethods 324:13-25, 2007). They can have potent antigen-binding capacityand can interact with novel epitopes that are inaccessible toconventional. V_(H)-V_(L) pairs. (Muyldermans et al., 2001). Alpacaserum IgG contains about 50% camelid heavy chain only IgG antibodies(HCAbs) (Maass et al., 2007). Alpacas may be immunized with knownantigens, such as TNF-α, and single domain antibodies can be isolatedthat bind to and neutralize the target antigen (Maass et al., 2007). PCRprimers that amplify virtually all alpaca antibody coding sequences havebeen identified and may be used to construct single domain phage displaylibraries, which can be used for antibody fragment isolation by standardbiopanning techniques well known in the art (Maass et al., 2007).

In certain embodiments, the sequences of antibodies or antibodyfragments, such as the Fc portions of antibodies, may be varied tooptimize their physiological characteristics, such as the half-life inserum. Methods of substituting amino acid sequences in proteins arewidely known in the art, such as by site-directed mutagenesis (e.g.Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed,1989). In preferred embodiments, the variation may involve the additionor removal of one or more glycosylation sites in the Fc sequence (e.g.,U.S. Pat. No. 6,254,868, the Examples section of which is incorporatedherein by reference). In other preferred embodiments, specific aminoacid substitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797).

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of, for example, a human antibody have been replaced by thevariable regions of, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. The affinity of humanized antibodies for a targetmay also be increased by selected modification of the CDR sequences(WO0029584A1). Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990).

Human Antibodies

In another embodiment, an antibody may be a human monoclonal antibody.Such antibodies may be obtained from transgenic mice that have beenengineered to produce specific human antibodies in response to antigenicchallenge. In this technique, elements of the human heavy and lightchain locus are introduced into strains of mice derived from embryonicstem cell lines that contain targeted disruptions of the endogenousheavy chain and light chain loci. The transgenic mice can synthesizehuman antibodies specific for human antigens, and the mice can be usedto produce human antibody-secreting hybridomas. Methods for obtaininghuman antibodies from transgenic mice are described by Green et al.,Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), andTaylor et al., Int. Immun. 6:579 (1994).

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as HIV infection or AIDS. The advantage toconstructing human antibodies from a diseased individual is that thecirculating antibody repertoire may be biased towards antibodies againstdisease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the and chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods, such as biopanning. The skilled artisanwill realize that this technique is exemplary only and any known methodfor making and screening human antibodies or antibody fragments by phagedisplay may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. A non-limiting example ofsuch a system is the XenoMouse® (e.g., Green et al., 1999, J. Immunol.Methods 231:11-23) from Abgenix (Fremont, Calif.). In the XenoMouse® andsimilar animals, the mouse antibody genes have been inactivated andreplaced by functional human antibody genes, while the remainder of themouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Such human antibodies may be coupled to other molecules bychemical cross-linking or other known methodologies. Transgenicallyproduced human antibodies have been shown to have therapeutic potential,while retaining the pharmacokinetic properties of normal humanantibodies (Green et al., 1999). The skilled artisan will realize thatthe claimed compositions and methods are not limited to use of theXenoMouse® system but may utilize any transgenic animal that has beengenetically engineered to produce human antibodies.

HIV Neutralizing Antibodies

In certain embodiments, neutralizing antibodies or fragments thereofthat are capable of inhibiting the infectivity of HIV are preferred. Avariety of HIV neutralizing antibodies are known in the art and any suchknown antibodies or fragments thereof may be used, including but notlimited to P4/D10, 2G12 (e.g., Joos et al., Antimicrob Agents Chemother2006, 50:1773-79), 4E10 (Joos et al., 2006), 2F5 (Joos et al., 2006),b12 (e.g., Wu et al., J Virol 2006, 80:2585), X5 (Moulard et al., ProcNatl Acad Sci 2002, 99:6913-18) or any combination thereof. Wheremultispecific antibodies or fragments are used, the skilled artisan willrealize that multiple antibodies or fragments that bind to the same ordifferent HIV epitopes may be combined. Although antibodies against theHIV envelope protein (gp120) and/or gp41 are preferred, the skilledartisan will realize that other HIV target antigens may be utilized todevelop antibodies or fragments thereof that will target HIV-infectedcells. In some cases, antibodies or fragments that bind to one or moreHIV antigens in combination with T-cell antigens (e.g., CD4, CCR5 and/orCXCR4) may be utilized.

Fusion Proteins

Various embodiments may concern fusion proteins. These moleculesgenerally have all or a substantial portion of a peptide, linked at theN- or C-terminus, to all or a portion of a second polypeptide orprotein. For example, fusions may employ leader sequences from otherspecies to permit the recombinant expression of a protein in aheterologous host. Another useful fusion includes the attachment of animmunologically active domain, such as an antibody or fragment, to atherapeutic agent, such as a peptide or protein toxin or enzyme. Yetanother useful form of fusion may include attachment of a moiety of usefor purification, such as the FLAG epitope (Prickett et al., 1989,Biotechniques 7:580-589; Castrucci et al., 1992, J Virol 66:4647-4653).Methods of generating fusion proteins are well known to those of skillin the art. Such proteins may be produced, for example, by chemicalattachment using bifunctional cross-linking reagents, by de novosynthesis of the complete fusion protein, or by attachment of a DNAsequence encoding a first protein or peptide to a DNA sequence encodinga second peptide or protein, followed by expression of the intact fusionprotein.

Immunoconjugates

In various embodiments, the anti-HIV antibodies, antibody fragments orother targeting molecules of the DNL complex may be directly conjugatedto one or more therapeutic agents. Exemplary therapeutic agents may beselected from the group consisting of cytotoxic agents, drugs, toxins,radionuclides, enzymes, hormones, cytokines or other immunomodulators.

Therapeutic agents of use may comprise one or more of aplidin,azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin,irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, daunomycin glucuronide,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyurea, idarubicin, ifosfamide, L-asparaginase, leucovorin,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, phenylbutyrate, procarbazine, pentostatin, PSI-341, semustine, streptozocin,taxanes, thioguanine, thiotepa, teniposide, topotecan, uracil mustard,velcade, vinblastine, vinorelbine, vincristine, ricin, abrin,ribonuclease, onconase, rapLR1, DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonasexotoxin, Pseudomonas endotoxin, an antisense oligonucleotide, aninterference RNA, or a combination thereof.

Conjugation can be via, for example, covalent attachments to amino acidresidues containing amine, carboxyl, thiol or hydroxyl groups in theirside-chains. Various conventional linkers may be used for this purpose,for example, diisocyanates, diisothiocyanates, bis(hydroxysuccinimide)esters, carbodiimides, maleimide-hydroxysuccinimide esters,glutaraldehyde and the like. Conjugation of agents to the HIV targetingmolecules preferably does not significantly affect the binding activityor specificity compared to the unmodified structures. In addition,cytotoxic and/or virostatic agents may be first coupled to a polymericcarrier, which is then conjugated to a HIV targeting molecule. For thismethod, see Ryser et al., Proc. Natl. Acad. Sci. USA, 75:3867-3870,1978, U.S. Pat. No. 4,699,784, and U.S. Pat. No. 4,046,722, which areincorporated herein by reference.

The conjugates described herein can be prepared by methods known forlinking antibodies with lipids, carbohydrates, proteins, radionuclides,or other atoms and molecules. For example, the HIV targeting moleculesdescribed herein can be linked to one or more of the carriers describedherein (e.g., lipids, polymers, liposomes, micelles, or nanoparticles)to form a conjugate, which can then incorporate a therapeutic ordiagnostic agent either covalently, non-covalently, or otherwise.Alternatively, any of the HIV targeting molecules described herein canbe conjugated directly with one or more therapeutic or diagnostic agentsdescribed herein.

For example, a HIV targeting molecule can be radiolabeled with ¹³¹I andconjugated to a lipid, such that the resulting conjugate can form aliposome. The liposome may incorporate one or more therapeutic (e.g., adrug such as FUdR-dO) or diagnostic agents. The formation of liposomesand micelles is known in the art. See, e.g., Wrobel and Collins,Biochimica et Biophysica Acta (1995), 1235: 296-304; Lundberg et al., J.Pharm. Pharmacol. (1999), 51:1099-1105; Lundberg et al., Int. J. Pharm.(2000), 205:101-108; Lundberg, J. Pharm. Sci. (1994), 83:72-75; Xu etal., Molec. Cancer Ther. (2002), 1:337-346; Torchilin et al., Proc.Nat'l. Acad. Sci., U.S.A. (2003), 100:6039-6044; U.S. Pat. No.5,565,215; U.S. Pat. No. 6,379,698; and U.S. 2003/0082154.

Nanoparticles or nanocapsules formed from polymers, silica, or metals,which are useful for drug delivery or imaging, have been described aswell. See, e.g., West et al., Applications of Nanotechnology toBiotechnology (2000), 11:215-217; U.S. Pat. No. 5,620,708; U.S. Pat. No.5,702,727; and U.S. Pat. No. 6,530,944. The conjugation of antibodies orbinding molecules to liposomes to form a targeted carrier fortherapeutic or diagnostic agents has been described. See, e.g., Bendas,Biodrugs (2001), 15:215-224; Xu et al., Mol. Cancer Ther (2002),1:337-346; Torchilin et al., Proc. Nat'l. Acad. Sci. U.S.A (2003),100:6039-6044; Bally, et al., J. Liposome Res. (1998), 8:299-335;Lundberg, Int. J. Pharm. (1994), 109:73-81; Lundberg, J. Pharm.Pharmacol. (1997), 49:16-21; Lundberg, Anti-cancer Drug Design (1998),13: 453-461. See also U.S. Pat. No. 6,306,393; U.S. Ser. No. 10/350,096;U.S. Ser. No. 09/590,284, and U.S. Ser. No. 60/138,284, filed Jun. 9,1999. All these references are incorporated herein by reference.

A wide variety of diagnostic and therapeutic agents can beadvantageously used to form the conjugates of the HIV targetingmolecules, or may be linked to haptens that bind to a recognition siteon the HIV targeting molecules. Diagnostic agents may includeradioisotopes, enhancing agents for use in MRI or contrast agents forultrasound imaging, and fluorescent compounds. Many appropriate imagingagents are known in the art, as are methods for their attachment toproteins or peptides (see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509,both incorporated herein by reference). Certain attachment methodsinvolve the use of a metal chelate complex employing, for example, anorganic chelating agent such a DTPA attached to the protein or peptide(U.S. Pat. No. 4,472,509).

In order to load a HIV targeting molecule with radioactive metals orparamagnetic ions, it may be necessary to first react it with a carrierto which multiple copies of a chelating group for binding theradioactive metals or paramagnetic ions have been attached. Such acarrier can be a polylysine, polysaccharide, or a derivatized orderivatizable polymeric substance having pendant groups to which can bebound chelating groups such as, e.g., ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins,polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and thelike known to be useful for this purpose. Carriers containing chelatesare coupled to the HIV targeting molecule using standard chemistries ina way to minimize aggregation and loss of immunoreactivity.

Other, more unusual, methods and reagents that may be applied forpreparing such conjugates are disclosed in U.S. Pat. No. 4,824,659,which is incorporated herein in its entirety by reference. Particularlyuseful metal-chelate combinations include 2-benzyl-DTPA and itsmonomethyl and cyclohexyl analogs, used with diagnostic isotopes in thegeneral energy range of 60 to 4,000 keV. Some useful diagnostic nuclidesmay include ¹⁸F, ⁵²Fe, ⁶²Ca, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc,^(94m)Tc, ^(99m)Tc, or ¹¹¹In. The same chelates complexed withnon-radioactive metals, such as manganese, iron and gadolinium, areuseful for MRI, when used along with the HIV targeting molecules andcarriers described herein. Macrocyclic chelates such as NOTA, DOTA, andTETA are of use with a variety of metals and radiometals, mostparticularly with radionuclides of gallium, yttrium and copper,respectively. Such metal-chelate complexes can be made very stable bytailoring the ring size to the metal of interest. Other ring-typechelates, such as macrocyclic polyethers for complexing ²²³Ra, may beused.

Therapeutic agents include, for example, chemotherapeutic drugs such asvinca alkaloids, anthracyclines, epipodophyllotoxins, taxanes,antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors,antimitotics, antiangiogenic and proapoptotic agents, particularlydoxorubicin, methotrexate, taxol, CPT-11, camptothecans, and others fromthese and other classes of cytotoxic agents. Other cytotoxic agentsinclude nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes,folic acid analogs, pyrimidine analogs, purine analogs, platinumcoordination complexes, and the like. Suitable cytotoxic agents aredescribed in REMINGTON′S PHARMACEUTICAL SCIENCES, 19th Ed. (MackPublishing Co. 1995), and in GOODMAN AND GILMAN′S THE PHARMACOLOGICALBASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as wellas revised editions of these publications. Other suitable cytotoxicagents, such as experimental drugs, are known to those of skill in theart, and may be conjugated to the HIV targeting molecules describedherein using methods that are known in the art.

Another class of therapeutic agents consists of radionuclides that emitα-particles (such as ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra, ²²⁵Ac),β-particles (such as ³²P, ³³P, ⁴⁷Se, ⁶⁷Cu, ⁶⁷Ga, ⁸⁹Sr, ⁹⁰Y, ¹¹¹Ag, ¹²⁵I,¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁶Dy, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re), orAuger electrons (such as ¹¹¹In, ¹²⁵I, ⁶⁷Ga, ¹⁹¹Os, ^(193m)Pt, ^(195m)Pt,^(195m)Hg). The HIV targeting molecules may be labeled with one or moreof the above radionuclides using methods as described for the diagnosticagents.

In certain embodiments, the therapeutic agents of use may comprise oneor more aggresome inhibitors. Aggresomes are large intracellularcomplexes that were thought to form in response to misfolded protein(see, e.g., Heath et al., J. Cell Biol. 153:449-55, 2001; Johnstone etal., J. Cell Biol. 143:1883-98, 1998; Wileman, Science 312:875-78,2006). More recently, it has been suggested that aggresomes may functionin the assembly of viral particles (Heath et al., 2001; Wileman, 2006).Aggresome inhibitors may therefore function to block or inhibit theformation of new infectious viral particles from cells infected with HIVor other viruses. A variety of aggresome inhibitors are known, such asALLN, nocodazole, colchicine and vinblastine (Johnston et al., 1998),other microtubule inhibitors (Gerdes and Katsanis, Hum. Molec. Genet.14:R291-300, 2005); bortezomib (VELCADE®) (Catley et al., Blood108:3441-49, 2006), tubacin, histone deacetylase inhibitors (Corcoran etal., Curr. Biol. 14:488-92, 2004), and any such known aggresomeinhibitor may be used.

In various embodiments, one or more immunomodulators may be conjugatedto an anti-HIV antibody or fragment. Alternatively, an immunomodulatormay be attached to an AD or DDD moiety for incorporation into a DNLcomplex, as described below. As used herein, the term “immunomodulator”includes cytokines, stem cell growth factors, lymphotoxins andhematopoietic factors, such as interleukins, colony stimulating factors,interferons (e.g., interferons-α, -β and -γ) and the stem cell growthfactor designated “Si factor.” Examples of suitable immunomodulatormoieties include IL-2, IL-6, IL-10, IL-12, IL-18, IL-21,interferon-gamma, TNF-alpha, and the like.

The term “cytokine” is a generic term for proteins or peptides releasedby one cell population which act on another cell as intercellularmediators. As used broadly herein, examples of cytokines includelymphokines, monokines, growth factors and traditional polypeptidehormones. Included among the cytokines are growth hormones such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; prostaglandin, fibroblast growth factor;prolactin; placental lactogen, OB protein; tumor necrosis factor-α and-β; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-21, LW, G-CSF, GM-CSF, M-CSF, EPO, kit-ligand or FLT-3,angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT.As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

Chemokines generally act as chemoattractants to recruit immune effectorcells to the site of chemokine expression. It may be advantageous toexpress a particular chemokine gene in combination with, for example, acytokine gene, to enhance the recruitment of other immune systemcomponents to a site of treatment. Chemokines include, but are notlimited to, RANTES, MCAF, MW1-alpha, MIP1-Beta, and W-10. The skilledartisan will recognize that certain cytokines are also known to havechemoattractant effects and could also be classified under the termchemokines. Similarly, the terms immunomodulator and cytokine overlap intheir respective members.

A suitable peptide containing a detectable label (e.g., a fluorescentmolecule), or a virostatic and/or cytotoxic agent, (e.g., aradioiodine), can be covalently, non-covalently, or otherwise associatedwith the HIV targeting molecules. For example, a therapeutically usefulconjugate can be obtained by incorporating a photoactive agent or dyeonto the HIV targeting molecules. Fluorescent compositions, such asfluorochrome, and other chromogens, or dyes, such as porphyrinssensitive to visible light, have been used to detect and to treatlesions by directing the suitable light to the lesion. In therapy, thishas been termed photoradiation, phototherapy, or photodynamic therapy.See Joni et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHERDISEASES (Libreria Progetto 1985); van den Bergh, Chem. Britain (1986),22:430. Moreover, monoclonal antibodies have been coupled withphotoactivated dyes for achieving phototherapy. See Mew et al., J.Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380; Oseroffet al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem., Photochem.Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol. Res. (1989),288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422; Pelegrin etal., Cancer (1991), 67:2529.

HIV Fusion Inhibitors

HIV fusion inhibitors are described in PCT Patent Application Publ. No.WO 2007045463, the entire text of which is incorporated herein byreference. Generally, infection of cells by human immunodeficiency virus(HIV) is effected by a process in which the membrane of the cells to beinfected and the viral membrane are fused. The viral envelopeglycoprotein complex (gp120/gp41) interacts with a cell surface receptorlocated on the membrane of the cell to be infected. The binding of gp120to e.g. the CD4 receptor in combination with a co-receptor such as CCR-5or CXCR-4, causes a change in the conformation of the gp120/gp41complex. As a result of this conformational change the gp41 protein isable to insert into the membrane of the target cell. This insertion isthe beginning of the membrane fusion process.

It is known that the amino acid sequence of the gp41 protein differsbetween the different HIV strains because of naturally occurringpolymorphisms. But the same domain architecture can be recognized, afusion signal, two heptad repeat domains (HR1, HR2) and a transmembranedomain. The fusion (or fusogenic) domain participates in the insertioninto and disintegration of the cell membrane. Peptides with amino acidsequences deduced from the HR1 or HR2 domain of gp41 are effective invitro and in vivo inhibitors of HIV uptake into cells (see, e.g. U.S.Pat. Nos. 5,464,933; 5,656,480; 6,258,782; 6,348,568; 6,656,906). Forexample, T20, an HR2 peptide and T651 (U.S. Pat. No. 6,479,055) arepotent inhibitors of HIV infection. Attempts have been made to enhancethe efficacy of HR2 derived peptides, for example by amino acidsubstitution or chemical crosslinking (Sia et al, 2002, PNAS USA99:14664-14669; Otaka et al, 2002, Angew. Chem. Int. 41:2937-2940).

Exemplary anti-fusogenic peptides are found in U.S. Pat. Nos. 5,464,933;5,656,480; 6,013,263; 6,017,536; 6,020,459; 6,093,794; 6,060,065;6,258,782; 6,348,568; 6,479,055; 6,656,906; and PCT Patent ApplicationPubl. Nos. WO 1996/19495, WO 1996/40191, WO 1999/59615, WO 2000/69902,and WO 2005/067960, the Examples section of each incorporated herein byreference. The skilled artisan will realize that any such HIV fusioninhibitor may be incorporated into the subject DNL complexes utilizingthe techniques described in the Examples below.

Interference RNA

In certain embodiments a DNL complex may be utilized to deliver an siRNAor interference RNA species. The siRNA, interference RNA or therapeuticgene may be attached to a carrier moiety that is incorporated into a DNLconstruct. A variety of carrier moieties for siRNA have been reportedand any such known carrier may be used. Non-limiting examples ofcarriers include protamine (Rossi, 2005, Nat Biotech 23:682-84; Song etal., 2005, Nat Biotech 23:709-17); dendrimers such as PAMAM dendrimers(Pan et al., 2007, Cancer Res. 67:8156-8163); polyethylenimine(Schiffelers et al., 2004, Nucl Acids Res 32:e149); polypropyleneimine(Taratula et al., 2009, J Control Release 140:284-93); polylysine (Inoueet al., 2008, J Control Release 126:59-66); histidine-containingreducible polycations (Stevenson et al., 2008, J Control Release130:46-56); histone H1 protein (Haberland et al., 2009, Mol Biol Rep26:1083-93); cationic comb-type copolymers (Sato et al., 2007, J ControlRelease 122:209-16); polymeric micelles (U.S. Patent Application Publ.No. 20100121043); and chitosan-thiamine pyrophosphate (Rojanarata etal., 2008, Pharm Res 25:2807-14). The skilled artisan will realize thatin general, polycationic proteins or polymers are of use as siRNAcarriers. The skilled artisan will further realize that siRNA carrierscan also be used to carry other oligonucleotide or nucleic acid species,such as anti-sense oligonucleotides or short DNA genes.

Many siRNA species are commercially available from known sources, suchas Sigma-Aldrich (St Louis, Mo.), Invitrogen (Carlsbad, Calif.), SantaCruz Biotechnology (Santa Cruz, Calif.), Ambion (Austin, Tex.),Dharmacon (Thermo Scientific, Lafayette, Colo.), Promega (Madison,Wis.), Mirus Bio (Madison, Wis.) and Qiagen (Valencia, Calif.), amongmany others. Other publicly available sources of siRNA species includethe siRNAdb database at the Stockholm Bioinformatics Centre, theMIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the BroadInstitute, and the Probe database at NCBI. For example, there are 30,852siRNA species in the NCBI Probe database. The skilled artisan willrealize that for any gene of interest, either a siRNA species hasalready been designed, or one may readily be designed using publiclyavailable software tools. Other known siRNA species have been reported,for example, for IKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 andFlk-1/KDR (U.S. Pat. No. 7,148,342); Bc12 and EGFR (U.S. Pat. No.7,541,453); CDC20 (U.S. Pat. No. 7,550,572); transducin (beta)-like 3(U.S. Pat. No. 7,576,196); KRAS (U.S. Pat. No. 7,576,197); carbonicanhydrase II (U.S. Pat. No. 7,579,457); complement component 3 (U.S.Pat. No. 7,582,746); interleukin-1 receptor-associated kinase 4 (IRAK4)(U.S. Pat. No. 7,592,443); survivin (U.S. Pat. No. 7,608,7070);superoxide dismutase 1 (U.S. Pat. No. 7,632,938); MET proto-oncogene(U.S. Pat. No. 7,632,939); IGF-1R (U.S. Pat. No. 7,638,621); ICAM1 (U.S.Pat. No. 7,642,349); complement factor B (U.S. Pat. No. 7,696,344); p53(7,781,575), and apolipoprotein B (7,795,421). Such known siRNA speciesmay be delivered using the subject DNL complexes.

Immunotoxins Comprising Ranpirnase (Rap)

Ribonucleases, in particular, Rap (Lee, Exp Opin Biol Ther 2008;8:813-27) and its more basic variant, amphinase (Ardelt et al., CurrPharm Biotechnol 2008:9:215-25), are potential cytotoxic agents (Lee andRaines, Biodrugs 2008; 22:53-8). Rap is a single-chain ribonuclease of104 amino acids originally isolated from the oocytes of Rana pipiens.Rap exhibits cytostatic and cytotoxic effects on a variety of cell linesin vitro, as well as antitumor activity in vivo. The amphibianribonuclease enters cells via receptor-mediated endocytosis and onceinternalized into the cytosol, selectively degrades tRNA, resulting ininhibition of protein synthesis and induction of apoptosis. Rap can beadministered repeatedly to patients without an untoward immune response,with reversible renal toxicity reported to be dose-limiting (Mikulski etal., J Clin Oncol 2002; 20:274-81; Int J Oncol 1993; 3:57-64).

Conjugation or fusion of Rap to a targeting antibody or antibodyfragment is a promising approach to enhance its potency, as firstdemonstrated for LL2-onconase (Newton et al., Blood 2001; 97:528-35), achemical conjugate comprising Rap and a murine anti-CD22 monoclonalantibody (MAb), and subsequently for 2L-Rap-hLL1-γ4P, a fusion proteincomprising Rap and a humanized anti-CD74 MAb (Stein et al., Blood 2004;104:3705-11).

The method used to generate 2L-Rap-hLL1-γ4P allowed us to develop aseries of structurally similar immunotoxins, referred to in general as2L-Rap-X, all of which consist of two Rap molecules, each connected viaa flexible linker to the N-terminus of one L chain of an antibody ofinterest (X). We have also generated another series of immunotoxins ofthe same design, referred to as 2LRap(Q)-X, by substituting Rap with itsnon-glycosylation form of Rap, designated as Rap(Q) to denote that thepotential glycosylation site at Asn69 is changed to Gln (or Q, singleletter code). For both series, we made the IgG as either IgG1(γ1) orIgG4(γ4), and to prevent the formation of IgG4 half molecules (Aalberseand Schuurman, Immunology 2002; 105:9-19), we converted the serineresidue in the hinge region (S228) of IgG4 to proline (γ4P). Apyroglutamate residue at the N-terminus of Rap is required for the RNaseto be fully functional (Liao et al., Nucleic Acids Res 2003;31:5247-55).

The skilled artisan will recognize that the cytotoxic RNase moietiessuitable for use in the present invention include polypeptides having anative ranpirnase structure and all enzymatically active variantsthereof. These molecules advantageously have an N-terminal pyroglutamicacid resides that appears essential for RNase activity and are notsubstantially inhibited by mammalian RNase inhibitors. Nucleic acid thatencodes a native cytotoxic RNase may be prepared by cloning andrestriction of appropriate sequences, or using DNA amplification withpolymerase chain reaction (PCR). The amino acid sequence of Rana Pipiensranpirnase can be obtained from Ardelt et al., J. Biol. Chem., 256: 245(1991), and cDNA sequences encoding native ranpirnase, or aconservatively modified variation thereof, can be gene-synthesized bymethods similar to the en bloc V-gene assembly method used in hLL2humanization. (Leung et al., Mol. Immunol., 32: 1413, 1995). Methods ofmaking cytotoxic RNase variants are known in the art and are within theskill of the routineer.

As described in the Examples below, Rap conjugates of targetingantibodies may be made using the DNL technology. The DNL Rap-antibodyconstructs show potent cytotoxic activity that can be targeted todisease-associated cells.

Formulation and Administration

The DNL constructs may be further formulated to obtain compositions thatinclude one or more pharmaceutically suitable excipients, one or moreadditional ingredients, or some combination of these. These can beaccomplished by known methods to prepare pharmaceutically usefuldosages, whereby the active ingredients are combined in a mixture withone or more pharmaceutically suitable excipients. Sterilephosphate-buffered saline is one example of a pharmaceutically suitableexcipient. Other suitable excipients are well known to those in the art.See, e.g., Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERYSYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON′S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

One route for administration of the compositions described herein isparenteral injection. In parenteral administration, the compositionswill be formulated in a unit dosage injectable form such as a solution,suspension or emulsion, in association with a pharmaceuticallyacceptable excipient. Such excipients are inherently nontoxic andnontherapeutic. Examples of such excipients are saline, Ringer'ssolution, dextrose solution and Hank's solution. Nonaqueous excipientssuch as fixed oils and ethyl oleate may also be used. A preferredexcipient is 5% dextrose in saline. The excipient may contain minoramounts of additives such as substances that enhance isotonicity andchemical stability, including buffers and preservatives. Other methodsof administration, including oral administration, are also contemplated.

Formulated compositions comprising DNL complexes can be used forintravenous administration via, for example, bolus injection orcontinuous infusion. Compositions for injection can be presented in unitdosage form, e.g., in ampules or in multi-dose containers, with an addedpreservative. Compositions can also take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the compositions can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compositions may be administered in solution. The pH of the solutionshould be in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5. Theformulation thereof should be in a solution having a suitablepharmaceutically acceptable buffer such as phosphate,tris(hydroxymethyl)aminomethane-HCl or citrate and the like. Bufferconcentrations should be in the range of 1 to 100 mM. The formulatedsolution may also contain a salt, such as sodium chloride or potassiumchloride in a concentration of 50 to 150 mM. An effective amount of astabilizing agent such as glycerol, albumin, a globulin, a detergent, agelatin, a protamine or a salt of protamine may also be included.Systemic administration of the formulated composition is typically madeevery two to three days or once a week if a humanized form of anti-HIVantibody is used. Usually administration is by either intramuscularinjection or intravascular infusion.

The compositions may be administered to subcutaneously or by otherparenteral routes. Moreover, the administration may be by continuousinfusion or by single or multiple boluses. Methods useful for theantibodies or immunoconjugates can be applied to the compositionsdescribed herein. In general, the dosage of an administeredimmunoconjugate, fusion protein or naked antibody for humans will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of the activeingredient that is in the range of from about 1 mg/kg to 20 mg/kg as asingle intravenous infusion, although a lower or higher dosage also maybe administered as circumstances dictate. This dosage may be repeated asneeded, for example, once per week for 4-10 weeks, preferably once perweek for 8 weeks, and more preferably, once per week for 4 weeks. It mayalso be given less frequently, such as every other week for severalmonths. The dosage may be given through various parenteral routes, withappropriate adjustment of the dose and schedule.

Pharmaceutical methods employed to control the duration of action ofimmunoconjugates or antibodies may be applied to the formulatedcompositions described herein. Control release preparations can beachieved through the use of biocompatible polymers to complex or adsorbthe immunoconjugate or naked antibody, for example, matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. See Sherwood et al.,Bio/Technology (1992), 10: 1446. The rate of release from such a matrixdepends upon the molecular weight of the DNL complex, the amount of DNLcomplex within the matrix, and the size of dispersed particles. SeeSaltzman et al., Biophys. J (1989), 55: 163; Sherwood et al., supra.Other solid dosage forms are described in Ansel et al., PHARMACEUTICALDOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18thEdition (Mack Publishing Company 1990), and revised editions thereof.

A DNL complex linked to a radionuclide may be effective for therapy.After it has been determined that the DNL complex is localized at one ormore infectious sites in a subject, higher doses of the labeledcomposition, generally from 20 mCi to 150 mCi per dose for ¹³¹I, 5 mCito 30 mCi per dose for ⁹⁰Y, or 5 mCi to 20 mCi per dose of ¹⁸⁶Re, eachbased on a 70 kg patient weight, are injected. Injection may beintravenous, intraarterial, intralymphatic, intrathecal, orintracavitary (i.e., parenterally), and may be repeated. It may beadvantageous for some therapies to administer multiple, divided doses,thus providing higher toxic doses without usually effecting aproportional increase in radiation of normal tissues.

Kits

Some embodiments concern kits for practicing the claimed methods. Thekit may include a DNL construct. The kit components may be packaged intocontainers, such as vials that contain sterile, lyophilized formulationsof a composition that are suitable for reconstitution. A kit may alsocontain one or more buffers suitable for reconstitution and/or dilutionof other reagents. Other containers that may be used include, but arenot limited to, a pouch, tray, box, tube, or the like. Kit componentsmay be packaged and maintained sterilely within the containers. Anothercomponent that can be included is instructions to a person using a kitfor its use.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the Examples which follow representtechniques discovered to function well in the practice of the invention,and thus can be considered to constitute preferred modes for itspractice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1 Inhibition of HIV Infection In Vitro and In Vivo UsingConjugated Anti-HIV Antibodies

Summary

Murine monoclonal antibody (MAb) against the envelope antigen of HIV(P4/D10) was conjugated with the conventional anti-cancer drug,doxorubicin, and tested against infectious virus and infected cells,both in vitro and in vivo. P4/D10 antibody was incubated with free virus(neutralization) or HIV-infected cells (inhibition) and the resultinginfection was measured by a p24 capture enzyme-linked immunosorbentassay. In an HIV-1/MuLV mouse challenge model the ability of theconjugate to inhibit infection in vivo was measured.

Doxorubicin-conjugated P4/D10 neutralized HIV-1_(IIIB) and eliminatedintercellular spread and HIV replication in infected Jurkat cells invitro. It also protected mice from challenge with HIV-1_(IIIB)/MuLV atan eight-fold lower concentration than needed for free antibody, whereasno effects were observed for free drug or irrelevant conjugate controls.These results demonstrate that doxorubicin was concentrated toHIV-infected cells by the P4/D10 antibody, significantly (p=0.0001)contributing to HIV elimination.

In this study, we conjugated doxorubicin, an anticancer anthracyclinewith known pharmacology, toxicology, and antitumor activity in patients,to a neutralizing and ADCC-mediating monoclonal antibody (MAb) developedagainst the HIV-1 outer envelope gp120 (third variable loop region).

The P4/D10 antibody conjugated to doxorubicin was tested in vitro forits efficacy in eliminating HIV-1-infected cells among non-infectedcells and in a mouse model by removing HIV-1/MuLV (murine leukemiavirus) infected syngeneic cells from the intraperitoneal cavity. Theanti-gp120 antibody, P4/D10, neutralizes HIV-1 virus and mediates ADCC(Broliden et al., 1990). It has also been used in its unconjugated formin a phase-I clinical trial for late-stage HIV-1 infected individuals,where it decreased HIV antigens for an extended period of time (Hinkulaet al., 1994). The present study was the first to examine thecombination of P4/D10 in a drug-conjugated form in a preclinical HIVmodel, in comparison to free MAb, free drug, and the irrelevantantibodies hRS7 (Stein et al., Int J Cancer 1993, 55:938-946) and hLL1Griffiths et al., Clin Cancer Res 2003, 9:6567-6571; Sapra et al., ClinCancer Res 2005, 11:5257-5264), that were conjugated similarly withdoxorubicin.

Materials and Methods

Antibodies and Drug Conjugation.

Conjugation of doxorubicin with the IgG1κ anti-gp120 antibody P4/D10(Broliden et al., 1990) and control antibodies, as well as thepreparation of the bifunctional doxorubicin hydrazone derivative with amaleimide group, were performed according to Griffiths et al. (2003).Briefly, antibodies P4/D10, hLL1 (humanized anti-CD74), and hRS7(humanized anti-EGP-1) in a final concentration of approximately 9mg/ml, were mildly reduced with DTT (dithiothreitol) in PBS (pH 7.5)containing 5 mM EDTA, using about 2.2 mM final DTT concentration,corresponding to a 38-fold molar excess of the reductant with respect tothe antibodies. The solutions were incubated at 37° C. for 40 min. Thereduced MAbs were purified on spin-columns of SEPHADEX® G50/80 in 50 mMsodium acetate buffer containing 150 mM NaCl and 2 mM EDTA (pH 5.3). Thenumber of thiol groups generated on the antibodies was determined byEllman's assay. For conjugation, mildly reduced antibodies at 6.5 mg/mlwere mixed with the bifunctional doxorubicin. The incubates were kept onice for 15 min, and purified on spin columns of G50/80 in 0.1 M sodiumacetate (pH 6.5), followed by passage through a short column ofBio-Beads SM2 (Bio-Rad, Hercules, Calif.) equilibrated in the samebuffer. The products were analyzed for doxorubicin/MAb substitutionratios by measuring absorbance.

A GMP-produced lot of IgG from HIV infected patients (HIVIgG) (Guay etal. AIDS 2002, 16:1391-1400) was used as positive control and sera fromHIV-negative individuals as negative controls. Free doxorubicin, as wellas the anticancer humanized MAbs LL1 and RS7, similarly conjugated withdoxorubicin, were included as controls for the conjugated P4/D10antibody.

HIV-1 Neutralization Assay.

Doxorubicin P4/D10, unlabelled P4/D10, HIV immunoglobulin (HIVIgG), andHIV-negative serum were mixed with the HIV-1 isolate HIV-1_(IIIB) (LAI)and incubated for 1 h at 37° C. before 50,000 Jurkat T-cells/well wereadded. After 1 h of incubation, the cells were washed with medium andnew complete medium added (200 μl/well). After 7 days of culture, theamount of p24 produced was measured by a p24 capture ELISA(enzyme-linked immunosorbent assay) and the percent inhibition of HIV-1p24 production was calculated.

HIV-1 Inhibition In Vitro.

Jurkat T-cells were infected with HIV-1_(IIIB) by mixing 5-10×10⁶ cellswith 100×TCID₅₀ HIV-1_(IIIB) and incubating for 1 h at 37° C. The cellswere washed in medium and incubated at 37° C. Every third day, mediumwas changed and supernatant checked for p24 production. When close to100% of the cells were infected, different proportions ofHIV-1_(IIIB)-infected cells were mixed with uninfected cells. The cellswere treated with serial dilutions of antibodies, serum, or freedoxorubicin from 100 to 0.00001 μg/ml. After seven days of culture at37° C., HIV-1 p24 inhibition was measured and supernatants from cellspreviously treated with 0.1-10 μg/ml of doxorubicin-P4/D10, unconjugatedP4/D10, and 0.05-0.5 mg/ml HIV-negative serum were collected andtransferred to fresh Jurkat T-cells to test if infectious HIV wasidentified by the p24 ELISA at days 3, 7, 10, 12, and 15 afterinitiation of the culture.

HIV-1/MuLV Challenge Model.

A human T-cell line, CEM-1B, with a genetically integrated MuLV genomewas infected with HIV-1_(IIIB), which led to the production ofpseudoviruses with the HIV-1 genome and the MuLV envelope (Adang et al.,PNAS USA 1999, 96:12749-753; Hinkula et al., Cells Tissues Organs 2004,177:169-184). These virus supernatants were used to infect splenocytesfrom C57Bl/6xDBA F1 K^(b/d) mice transgenic for HLA-A201. Isogenic micewere challenged with HIV-1_(IIIB)/MuLV infected splenocytes i.p. andwere immediately given conjugated antibodies, free antibodies or freedoxorubicin i.p. Ten days after challenge, mice were sacrificed andperitoneal cells collected. Peritoneal cells were pelleted and added to1×10⁶ HIV susceptible Jurkat T-cells or human PBMC grown in 24-wellplates. From these secondary cultures, supernatant was removed and freshmedium added every 3-4 days. The amount of infectious HIV recovered inthe supernatant was measured for 3 weeks by p24 ELISA.

Statistical Analysis.

To compare the in vitro HIV-1 neutralizing capacities of the anti-gp120MAbs and control antibodies, Student's t-test and the non-parametricKruskal-Wallis test were used. Statistical comparisons between thegroups of mice treated with different antibodies were performed usingthe nonparametric Mann-Whitney U and Kruskal-Wallis tests. A differencewas considered significant when a p-value of <0.05 was obtained. Anon-parametric one-way ANOVA test was performed using PRISM® version4.0a (GRAPHPAD® Software, San Diego, Calif.) and was used forcomparisons of HIV-1 isolation and p24 antigen positivity between thestudy groups.

Results

The number of thiol groups generated on the respective antibodies bymild reduction, as well as the doxorubicin/MAb substitution ratios inthe final purified conjugates, ranged between 8.8 (P4/D10, hRS7) and 9.4(hLL1), giving a ratio of approximately 9 drug molecules per IgG.High-pressure liquid chromatographic analyses showed that the conjugatesand the native MAbs possessed similar retention times, with zero tominimal aggregation (data not shown).

No significant difference in HIV-1 neutralizing capacity of free HIV-1virus (FIG. 1A) could be shown between the doxorubicin-conjugated P4/D10MAb and either unconjugated P4/D10 MAb or the HIVIgG antibodies.However, all anti HIV-1 specific antibodies were significantly betterthen the negative control serum (p=0.001) at neutralizing HIV-1_(IIIB).

When 3% HIV-1_(IIIB) infected Jurkat cells were mixed with 97%uninfected cells, doxorubicin-P4/D10 mediated a significantly (p=0.002)stronger inhibition of intercellular spread of HIV-1 infection than freeP4/D10, doxorubicin-conjugated control antibody, hLL1, or freedoxorubicin at a concentration of 0.5 or 0.05 μg/ml (FIG. 1B). Similarresults were seen at all other concentrations of infected and uninfectedcells. It was of particular interest that the intercellular spread ofinfection appeared to be inhibited even more potently than the effectobtained with doxorubicin-P4/D10 as a neutralizing agent. Also, noinfectious virus could be found in the cultures treated with high dosesof doxorubicin-P4/D10, since no p24 production was detected aftertransfer of supernatants from these cell cultures to uninfected Jurkatcells (data not shown). The significant difference in effect betweendoxorubicin-P4/D10 and unconjugated P4/D10 could not have been predictedfrom the results on neutralization of free HIV-1 virus (FIG. 1A).

To test the efficacy of doxorubicin-P4/D10 antibody in vivo, mice weregiven isogeneic HIV/MuLV-infected cells together with conjugatesintraperitoneally. Peritoneal cells were harvested 10 days later andinfectious HIV was demonstrated in all controls, similar to previousstudies (Hinkula et al., 2004). The doxorubicin-P4/D10 antibodyprotected mice completely against challenge with HIV-1 infected primarylymphoid cells (p=0.0001) (FIG. 2). No infectious HIV was recovered fromperitoneal cells after challenge and treatment with 100 μg ofdoxorubicin-P4/D10 antibody. When mice were treated with 100 μg ofunconjugated P4/D10 antibody, all were positive for p24 production.Complete protection by antibody alone was seen only when the dose wasincreased eight-fold, to 800 μg unconjugated P4/D10 per mouse. None ofthe doxorubicin-conjugated control antibodies (hLL1 or hRS7) providedany protection at doses of 100-200 μg, nor did doses of 100-400 μg offree doxorubicin.

SUMMARY

Doxorubicin-P4/D 10 was capable of eliminating HIV-1 infected cells invitro, as well as in an experimental in vivo challenge model. Theability of the unconjugated P4/D10 MAb to mediate ADCC against HIV-1infected target cells as well as neutralizing HIV-1 (Broliden et al.,1990; Hinkula et al., 1994) may enhance its efficacy as a drugimmunoconjugate in a non-toxic manner.

Similarly efficacious anti-HIV immunoconjugates may be incorporated intoDNL complexes, utilizing the compositions and methods disclosed in thefollowing Examples.

Example 2 Preparation of Dock-and-Lock (DNL) Constructs

DDD and AD Fusion Proteins

The DNL technique can be used to make dimers, trimers, tetramers,hexamers, etc. comprising virtually any antibody, antibody fragment,immunomodulator, cytokine, PEG moiety, toxin, or other effector moiety.For certain preferred embodiments, an anti-HIV antibody or antibodyfragment and an HIV inhibitor may be produced as fusion proteinscomprising either a dimerization and docking domain (DDD) or anchoringdomain (AD) sequence. Although in preferred embodiments the DDD and ADmoieties may be the effector moieties as fusion proteins, the skilledartisan will realize that other methods of conjugation exist, such aschemical cross-linking, click chemistry reaction, etc.

The technique is not limiting and any protein or peptide of use may beproduced as an AD or DDD fusion protein for incorporation into a DNLcomplex. Where chemical cross-linking is utilized, the AD and DDDconjugates may comprise any molecule that may be cross-linked to an ADor DDD sequence using any cross-linking technique known in the art.

Expression Vectors

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (V_(H) and V_(L)) sequences.Using molecular biology tools known to those skilled in the art, theseIgG expression vectors can be converted into Fab-DDD or Fab-ADexpression vectors.

To generate Fab-DDD expression vectors, the coding sequences for thehinge, CH2 and CH3 domains of the heavy chain were replaced with asequence encoding the first 4 residues of the hinge, a 14 residueGly-Ser linker and a DDD moiety, such as the first 44 residues of humanRIIα (referred to as DDD1, SEQ ID NO:1). To generate Fab-AD expressionvectors, the sequences for the hinge, CH2 and CH3 domains of IgG werereplaced with a sequence encoding the first 4 residues of the hinge, a15 residue Gly-Ser linker and an AD moiety, such as a 17 residuesynthetic AD called AKAP-IS (referred to as AD1, SEQ ID NO:3), which wasgenerated using bioinformatics and peptide array technology and shown tobind RIIα, dimers with a very high affinity (0.4 nM). See Alto, et al.Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50.

Two shuttle vectors were designed to facilitate the conversion ofIgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1 expression vectors, asdescribed below.

Preparation of CH1 Antibody Domain

The CH1 antibody domain was amplified by PCR using the pdHL2 plasmidvector as a template. The left PCR primer consisted of the upstream (5′)end of the CH1 domain and a SacII restriction endonuclease site, whichis 5′ of the CH1 coding sequence. The right primer consisted of thesequence coding for the first 4 residues of the hinge (PKSC, SEQ IDNO:85) followed by four glycines and a serine, with the final two codons(GS) comprising a Bam HI restriction site. The 410 bp PCR amplimer wascloned into the PGEMT® PCR cloning vector (PROMEGA®, Inc.) and cloneswere screened for inserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of DDD1 preceded by 11 residues of the linker peptide, with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 86) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRL REARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, whichoverlap by 30 base pairs on their 3′ ends, were synthesized and combinedto comprise the central 154 base pairs of the 174 bp DDD1 sequence. Theoligonucleotides were annealed and subjected to a primer extensionreaction with Taq polymerase. Following primer extension, the duplex wasamplified by PCR. The amplimer was cloned into PGEMT® and screened forinserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of AD1 preceded by 11 residues of the linker peptide with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

GSGGGGSGGGGSQIEYLAKQIVDNAIQQA (SEQ ID NO: 87)

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplimerwas cloned into the PGEMT® vector and screened for inserts in the T7(5′) orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from PGEMT®with BamHI and NotI restriction enzymes and then ligated into the samesites in CH1-PGEMT® to generate the shuttle vector CH1-DDD1-PGEMT®.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from PGEMT®with BamHI and NotI and then ligated into the same sites in CH1-PGEMT®to generate the shuttle vector CH1-AD1-PGEMT®.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacITIEagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective PGEMT® shuttle vector.

Construction of h679-Fd-AD1-pdHL2

h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fabwith AD1 coupled to the carboxyl terminal end of the CH1 domain of theFd via a flexible Gly/Ser peptide spacer composed of 14 amino acidresidues. A pdHL2-based vector containing the variable domains of h679was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/EagIfragment with the CH1-AD1 fragment, which was excised from theCH1-AD1-SV3 shuttle vector with SacII and EagI. The 679 antibody is ahapten-binding antibody specific for histamine succinyl glycine (HSG)(see, e.g., U.S. Pat. Nos. 7,429,381; 7,563,439).

Production and Purification of h679-Fab-AD1

The h679-Fd-AD1-pdHL2 vector was linearized by digestion with Sal Irestriction endonuclease and transfected into Sp/EEE myeloma cells byelectroporation. The di-cistronic expression vector directs thesynthesis and secretion of both h679 kappa light chain and h679 Fd-AD1,which combine to form h679 Fab-AD1. Following electroporation, the cellswere plated in 96-well tissue culture plates and transfectant cloneswere selected with 0.05 μM methotrexate (MTX). Clones were screened forprotein expression by ELISA using microtiter plates coated with aBSA-IMP260 (HSG) conjugate and detection with HRP-conjugated goatanti-human Fab. BIAcore analysis using an HSG (IMP239) sensorchip wasused to determine the productivity by measuring the initial slopeobtained from injection of diluted media samples. The highest producingclone had an initial productivity of approximately 30 mg/L. A total of230 mg of h679-Fab-AD1 was purified from 4.5 liters of roller bottleculture by single-step IMP291 affinity chromatography. Culture media wasconcentrated approximately 10-fold by ultrafiltration before loadingonto an IMP291-affigel column. The column was washed to baseline withPBS and h679-Fab-AD1 was eluted with 1 M imidazole, 1 mM EDTA, 0.1 MNaAc, pH 4.5. SE-HPLC analysis of the eluate shows a single sharp peakwith a retention time consistent with a 50 kDa protein (not shown). Onlytwo bands, which represent the polypeptide constituents of h679-AD1,were evident by reducing SDS-PAGE analysis (not shown).

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN-14(I)-pdHL2, which has been used to produce hMN-14 IgG, wasconverted to C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagIrestriction endonucleases to remove the CH1-CH3 domains and insertion ofthe CH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttlevector with SacII and EagI.

The same technique has been utilized to produce plasmids for Fabexpression of a wide variety of known antibodies, such as hLL1, hLL2,hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others. Generally,the antibody variable region coding sequences were present in a pdHL2expression vector and the expression vector was converted for productionof an AD- or DDD-fusion protein as described above. The AD- andDDD-fusion proteins comprising a Fab fragment of any of such antibodiesmay be combined, in an approximate ratio of two DDD-fusion proteins perone AD-fusion protein, to generate a trimeric DNL complex comprising twoFab fragments of a first antibody and one Fab fragment of a secondantibody.

Production and Purification of C-DDD1-Fab-hMN-14

The C-DDD1-Fd-hMN-14-pdHL2 vectors was transfected into Sp2/0-derivedmyeloma cells by electroporation. C-DDD1-Fd-hMN-14-pdHL2 is adi-cistronic expression vector, which directs the synthesis andsecretion of both hMN-14 kappa light chain and hMN-14 Fd-DDD1, whichcombine to form C-DDD1-hMN-14 Fab. The fusion protein forms a stablehomodimer via the interaction of the DDD 1 domain.

Following electroporation, the cells were plated in 96-well tissueculture plates and transfectant clones were selected with 0.05 μMmethotrexate (MTX). Clones were screened for protein expression by ELISAusing microtiter plates coated with WI2 (a rat anti-id monoclonalantibody to hMN-14) and detection with HRP-conjugated goat anti-humanFab. The initial productivity of the highest producing C-DDD1-Fab-hMN14Fab clone was 60 mg/L. The secreted C-DDD1-Fab-hMN14 may be purified byaffinity column chromatograph using and AD1 column. AD1-C is a syntheticpeptide consisting of the AD1 sequence and a carboxyl terminal cysteineresidue, which was used to couple the peptide to Affigel followingreaction of the sulfhydryl group with chloroacetic anhydride.DDD-containing dimer structures specifically bind to the AD1-C-Affigelresin at neutral pH and can be eluted at low pH (e.g., pH 2.5).

The binding activity of C-DDD1-Fab-hMN-14 was determined by SE-HPLCanalysis of samples in which the test article was mixed with variousamounts of WI2. A sample prepared by mixing WI2 Fab andC-DDD1-Fab-hMN-14 at a molar ratio of 0.75:1 showed three peaks, whichwere attributed to unbound C-DDD1-Fab-hMN14 (8.71 min),C-DDD1-Fab-hMN-14 bound to one WI2 Fab (7.95 min), and C-DDD1-Fab-hMN14bound to two WI2 Fabs (7.37 min) (not shown). When a sample containingWI2 Fab and C-DDD1-Fab-hMN-14 at a molar ratio of 4 was analyzed, only asingle peak at 7.36 minutes was observed (not shown). These resultsdemonstrated that hMN14-Fab-DDD1 is dimeric and has two active bindingsites. A competitive ELISA demonstrated that C-DDD1-Fab-hMN-14 binds toCEA with an avidity similar to hMN-14 IgG, and significantly strongerthan monovalent hMN-14 Fab (not shown).

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 (SEQ ID NO:2) appended to the carboxyl terminus of theFd of hMN-14 via a 14 amino acid residue Gly/Ser peptide linker. Thefusion protein secreted is composed of two identical copies of hMN-14Fab held together by non-covalent interaction of the DDD2 domains.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide and residues 1-13 of DDD2, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

The duplex DNA was ligated with the shuttle vector CH1-DDD1-PGEMT®,which was prepared by digestion with BamHI and PstI, to generate theshuttle vector CH1-DDD2-PGEMT®. A 507 bp fragment was excised fromCH1-DDD2-PGEMT® with SacII and EagI and ligated with the IgG expressionvector hMN-14(I)-pdHL2, which was prepared by digestion with SacII andEagI. The final expression construct was designatedC-DDD2-Fd-hMN-14-pdHL2. Similar techniques have been utilized togenerated DDD2-fusion proteins of the Fab fragments of a number ofdifferent humanized antibodies.

h679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair to C-DDD2-Fab-hMN-14.h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchoring domain sequence of AD2 (SEQID NO:4) appended to the carboxyl terminal end of the CH1 domain via a14 amino acid residue Gly/Ser peptide linker. AD2 has one cysteineresidue preceding and another one following the anchor domain sequenceof AD1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-PGEMT®. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Creation of C-H-AD2-IgG-pdHL2 expression vectors

A plasmid shuttle vector was produced to facilitate the conversion ofany IgG-pdHL2 vector into a C-H-AD2-IgG-pdHL2 vector. The gene for theFc (CH2 and CH3 domains) was amplified using the pdHL2 vector as atemplate and Fc BglII Left and Fc Bam-EcoRI Right primers. The amplimerwas cloned in the PGEMT® PCR cloning vector. The Fc insert fragment wasexcised from PGEMT® with XbaI and BamHI restriction enzymes and ligatedwith AD2-pdHL2 vector that was prepared by digestion ofh679-Fab-AD2-pdHL2 with XbaI and BamHI, to generate the shuttle vectorFc-AD2-pdHL2.

Fc BgIII Left (SEQ ID NO: 88) 5′-AGATCTGGCGCACCTGAACTCCTG-3′Fc Bam-EcoRI Right (SEQ ID NO: 89)5′-GAATTCGGATCCTTTACCCGGAGACAGGGAGAG-3′

To convert any IgG-pdHL2 expression vector to a C-H-AD2-IgG-pdHL2expression vector, an 861 bp BsrGI/NdeI restriction fragment is excisedfrom the former and replaced with a 952 bp BsrGI/NdeI restrictionfragment excised from the Fc-AD2-pdHL2 vector. BsrGI cuts in the CH3domain and NdeI cuts downstream (3′) of the expression cassette.

Example 3 Generation of TF2 DNL Complex

A trimeric DNL complex designated TF2 was obtained by reactingC-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generatedwith >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. Thetotal protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.Subsequent steps involved TCEP reduction, HIC chromatography, DMSOoxidation, and IMP291 affinity chromatography. Before the addition ofTCEP, SE-HPLC did not show any evidence of a₂b formation (not shown).Addition of 5 mM TCEP rapidly resulted in the formation of a₂b complexconsistent with a 157 kDa protein expected for the binary structure (notshown). TF2 was purified to near homogeneity by IMP291 affinitychromatography (not shown). IMP291 is a synthetic peptide containing theHSG hapten to which the 679 Fab binds (Rossi et al., 2005, Clin CancerRes 11:7122s-29s). SE-HPLC analysis of the IMP291 unbound fractiondemonstrated the removal of a₄, a₂ and free kappa chains from theproduct (not shown).

The functionality of TF2 was determined by BIACORE® assay. TF2,C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent a₂bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 μg/ml (total protein)and passed over a sensorchip immobilized with HSG. The response for TF2was approximately two-fold that of the two control samples, indicatingthat only the h679-Fab-AD component in the control samples would bind toand remain on the sensorchip. Subsequent injections of WI2 IgG, ananti-idiotype antibody for hMN-14, demonstrated that only TF2 had aDDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD asindicated by an additional signal response (not shown). The additionalincrease of response units resulting from the binding of WI2 to TF2immobilized on the sensorchip corresponded to two fully functionalbinding sites, each contributed by one subunit of C-DDD2-Fab-hMN-14.This was confirmed by the ability of TF2 to bind two Fab fragments ofWI2 (not shown).

Serum Stability of TF2

The stability of TF2 in human sera was assessed using BIACORE®. TF2 wasdiluted to 0.1 mg/ml in fresh human serum and incubated at 37° C. under5% CO₂ for seven days. Daily samples were diluted 1:25 and then analyzedby BIACORE® using an IMP239 HSG sensorchip. An injection of WI2 IgG wasused to quantify the amount of intact and fully active TF2. Serumsamples were compared to control samples that were diluted directly fromthe stock. TF2 was highly stable in serum, retaining 98% of itsbispecific binding activity after 7 days (not shown).

Example 4 Production of AD- and DDD-linked Fab and IgG Fusion Proteinsfrom Multiple Antibodies

Using the techniques described in the preceding Examples, the IgG andFab fusion proteins shown in Table 5 were constructed and incorporatedinto DNL complexes. The fusion proteins retained the antigen-bindingcharacteristics of the parent antibodies and the DNL complexes exhibitedthe antigen-binding activities of the incorporated antibodies orantibody fragments.

TABLE 5 Fusion proteins comprising IgG or Fab Fusion Protein BindingSpecificity C-AD1-Fab-h679 HSG C-AD2-Fab-h679 HSG C-(AD)₂-Fab-h679 HSGC-AD2-Fab-h734 Indium-DTPA C-AD2-Fab-hA20 CD20 C-AD2-Fab-hA20L CD20C-AD2-Fab-hL243 HLA-DR C-AD2-Fab-hLL2 CD22 N-AD2-Fab-hLL2 CD22C-AD2-IgG-hMN-14 CEACAM5 C-AD2-IgG-hR1 IGF-1R C-AD2-IgG-hRS7 EGP-1C-AD2-IgG-hPAM4 MUC C-AD2-IgG-hLL1 CD74 C-DDD1-Fab-hMN-14 CEACAM5C-DDD2-Fab-hMN-14 CEACAM5 C-DDD2-Fab-h679 HSG C-DDD2-Fab-hA19 CD19C-DDD2-Fab-hA20 CD20 C-DDD2-Fab-hAFP AFP C-DDD2-Fab-hL243 HLA-DRC-DDD2-Fab-hLL1 CD74 C-DDD2-Fab-hLL2 CD22 C-DDD2-Fab-hMN-3 CEACAM6C-DDD2-Fab-hMN-15 CEACAM6 C-DDD2-Fab-hPAM4 MUC C-DDD2-Fab-hR1 IGF-1RC-DDD2-Fab-hRS7 EGP-1 N-DDD2-Fab-hMN-14 CEACAM5

The skilled artisan will realize that the DNL technique may be appliedto produce multimeric complexes comprising any combination ofantibodies, antibody fragments and/or other therapeutic agents, such asanti-HIV therapeutic agents. The Examples herein demonstrate thatantibodies or fragments thereof may be incorporated into DNL complexeswithout any impairment of the antibody binding characteristics, comparedto the parent antibodies.

Example 5 Production and Use of Multivalent DNL Complexes

Multivalent antibodies, either monospecific or bispecific, may improvethe efficacy of current therapeutic interventions involving a singlemonoclonal antibody (mAb). Multivalent anti-CD20 antibody antibodieswere generated from veltuzumab (hA20, see U.S. Pat. Nos. 7,151,164;7,435,803; 7,919,273). We applied the DNL method to prepare ahexavalent, anti-CD20 antibody, designated Hex-hA20, which comprises sixFabs with one Fc. We showed that Hex-hA20 retained the binding activityof all six Fabs, associated with CD20 in lipid rafts, affectedantibody-dependent cell-mediated cytotoxicity, but notcomplement-dependent cytotoxicity, and inhibited proliferation of Daudi,Raji, and Ramos cells in vitro at subnanomolar concentrations withoutthe need for a cross-linking antibody (Rossi et al., 2008, Cancer Res68:8384-92). In addition, Hex-hA20 induced strong homotypical adhesionand was inefficient in stimulating calcium mobilization (Id.) Thus,Hex-hA20 exhibited biological properties attributable to both type I andtype II anti-CD20 mAbs, as exemplified by rituximab and tositumomab,respectively. Although Hex-hA20 has a short serum half-life, it showedantitumor efficacy in tumor-bearing mice comparable with veltuzumab atequivalent doses (Id.)

The DNL method was also applied to generate two other multivalentanti-CD20 antibodies without the Fc region, Tri-hA20 and Tetra-hA20,comprising three and four Fabs of veltuzumab, respectively. Similar toHex-hA20, these were purified to near homogeneity and shown to havepotent antiproliferative activity in vitro (Id.), thus indicating theneed for clustering three or more CD20 molecules on the cell surface toinduce growth inhibition.

Materials and Methods

Cell Lines.

Daudi, Raji, and Ramos were purchased from the American Type CultureCollection. Sp/ESF, a variant of Sp2/0-Ag14 engineered to grow inserum-free medium, was used as the host cell for transfection.

Generation of Hex-hA20.

The expression vector encoding C_(H1)-DDD2-Fab-hA20 was generated fromthe C_(H3)-AD2-IgG-hA20-pdHL2 by excising the coding sequence for theC_(H1)-Hinge-C_(H2)-C_(H3) domains with SacII and EagI and replacing itwith a 507-bp sequence encoding C_(H1)-DDD2, which was excised from theC-DDD2-hMN-14-pdHL2 expression vector (as described in Example 2 above)with the same enzymes. The expression vector C_(H3)-AD2-IgG-hA20-pdHL2or C_(H1)-DDD2-Fab-hA20-pdHL2, each 30 μg, was linearized by digestionwith SalI and transfected into Sp/ESF (2.8×10⁶ cells) by electroporation(450 V, 25 μF). The pdHL2 vector contains the gene for dihydrofolatereductase, thus allowing clonal selection, as well as geneamplification, with methotrexate (MTX). After transfection, the cellswere plated in 96-well plates and selected in media containing 0.2 wonMTX. Clones were screened for C_(H3)-AD2-IgG-hA20 orC_(H1)-DDD2-Fab-hA20 productivity by a sandwich ELISA using 96-wellmicrotiter plates coated with WR2 (rat anti-idiotype antibody toveltuzumab) to capture the fusion protein, which was detected withhorseradish peroxidase-conjugated goat anti-human IgG F(ab′)₂. Wellsgiving the highest signal were expanded and ultimately used forproduction.

C_(H3)-AD2-IgG-hA20 and C_(H1)-DDD2-Fab-hA20 were produced in rollerbottles, purified by affinity chromatography on Protein A and Protein L,respectively, and stored in PBS. To generate Hex-hA20, a mixture ofC_(H1)-DDD2-Fab-hA20 (134 mg) and C_(H3)-AD2-IgG-hA20 (100 mg) wastreated with 1 mmol/L reduced glutathione at room temperature for 16 h,followed by 2 mmol/L oxidized glutathione for 24 h, from which Hex-hA20was purified by Protein A. DNL-20/14 was made similarly by reactingC_(H3)-AD2-IgG-hA20 with C_(m)-DDD2-Fab-hMN-14.

Generation of Tetra-hA20 and Tri-hA20.

Tetra-hA20 was obtained by purifying the tetrameric form ofC_(H1)-DDD2-Fab-hA20 over a SUPERDEX™-200 column. Tri-hA20 was obtainedby linking the dimeric form of C_(H1)-DDD2-Fab-hA20 covalently toC_(H1)-AD2-Fab-hA20, which was produced as described in Example 2 abovefor h679-Fab-AD2.

Competition ELISA.

Microtiter plates were coated overnight with veltuzumab at 5 μg/mL andblocked with PBS containing 2% bovine serum albumin (BSA) for 1 h.Hex-hA20 and veltuzumab, serially diluted in triplicate, were each mixedwith WR2 at 1 mmol/L and added to the coated wells. The bound WR2 wasquantified with peroxidase-conjugated goat anti-rat IgG andO-phenylenediamine dihydrochloride.

Flow Cytometry.

Apoptosis, viable cell counting, cell binding, and off-rate measurementswere performed by flow cytometry on a GUAVA® PCA (Guava Technologies,Inc.) using the manufacturer's reagents, protocols, and software.

Scatchard Analysis.

The maximum number of binding sites per cell and the apparent affinityconstants were determined by nonlinear regression analysis of thesaturation binding data obtained with radio-iodinated samples and Rajicells using PRISM® software (GRAPHPAD® Software, Inc.). Samples were runin triplicate. Immunoreactivity of each radiolabeled preparation was 90%or greater, as measured by binding to WR2.

Cell Proliferation Assay.

The in vitro cytotoxicity was determined using3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS), which is chemically reduced by metabolically active cells intoformazan, and the intensity of the resulting color is proportional tothe number of living cells.

CDC.

Cells were seeded in black 96-well microtitre plates at 5×10⁴ cells in50 μL/well and incubated with serial dilutions (concentration range,3.33×10⁻⁸ to 2.6×10⁻¹⁰ mol/L) of test and control mAbs in the presenceof human complement (1:20 final dilution) for 2 h at 37° C. and 5% CO₂.Viable cells were then quantified using the VYBRANT™ Cell MetabolicAssay Resazurin kit (Invitrogen). Controls included cells treated with0.25% Triton X-100 (100% lysis) and cells treated with complement alone(background).

ADCC.

Daudi cells were incubated with each test article in triplicate at 5μg/mL for 30 min at 37° C. and 5% CO₂. Freshly isolated peripheral bloodmononuclear cells obtained from healthy volunteers were then added at apredetermined optimal effector to target ratio of 50:1. After a 4-hincubation, cell lysis was assessed by CYTOTOX-ONE™ (Promega).

Calcium Mobilization.

Intracellular calcium was measured in Ramos cells loaded with 20 μmol/LFluo-3 AM (Invitrogen) using a Becton Dickinson FACSCAN® and the FlowJoprogram (Tree Star, Inc.). For all samples, a baseline was obtained for60 s before adding each test article, which includes ionomycin andanti-human IgM as positive controls. To evaluate the effect ofcross-linking, cells were incubated with 1 μg/mL of veltuzumab,rituximab, or tositumomab for further 15 min and stimulated with anappropriate second antibody (50 μg/mL final).

Homotypical Adhesion.

Daudi cells (1.5×10⁶/mL) were treated with veltuzumab, Tri-hA20,Tetra-hA20, or Hex-hA20 at 1 nmol/L for 20 h and then examined with aninverted phase-contrast microscope. The results were scoredsemiquantitatively according to Polyak and Deans (2002, Blood99:3256-62).

Animal Studies.

The pharmacokinetic analysis was performed in naive female Swiss-Webstermice and compared with Hex-hA20 and veltuzumab given either i.v. or s.c.using radio-iodinated samples. The in vivo efficacy was evaluated intumor-bearing SCID mice. Depletion of natural killer (NK) cells andneutrophils was performed as described (Hernandez-Ilizaliturri et al.,2003, Clin Cancer Res 9:5866-73), with the following modifications.Briefly, mice received i.p. injections of antimouse Gr-1 ascites (100μL) and TMβ-1 mAb (100 μg) 1 d before inoculating Raji cells and threemore weekly i.p. injections of antimouse Gr-1 ascites on days 6, 13, and20 to maintain neutrophil depletion, which was confirmed byfluorescence-activated cell sorting analysis of blood samples taken fromone treated and one untreated mouse on days 3, 13, and 20. Mice deemedto have succumbed to disease progression, when hind-limb paralysisdeveloped or if they otherwise became moribund, were humanelysacrificed. Additionally, if mice lost >20% of initial body weight, theywere sacrificed. Survival curves were analyzed using Kaplan-Meier plots(log-rank analysis) and PRISM® software.

Results

Hexavalent Antibodies Made by DNL.

Hex-hA20 was readily obtained by mixing C_(H1)-DDD2-Fab-hA20 andC_(H3)-AD2-IgG-hA20 under redox conditions followed by purification withProtein A. Both C_(H1)-DDD2-Fab-hA20 and C_(H3)-AD2-IgG-hA20 wereproduced with good yields as fusion proteins in myeloma cells, withsubsequent purification from culture supernatants by Protein L andProtein A, respectively.

The purity of Hex-hA20 by reducing SDS-PAGE showed only three bands fromthe constitutive polypeptides (data not shown). Nonreducing SDS-PAGEanalysis of Hex-hA20 confirmed its covalent structure, because no bandscorresponding to the monomeric form of C_(H3)-AD2-IgG-hA20 were observed(not shown). The molecular mass of Hex-hA20 was determined to be 368,475Da by MALDI-TOF mass spectrometry, which agrees well with the calculatedmolecular weight of 362 kDa for Hex-hA20 from the deduced amino acidsequences of the constituent polypeptides.

Binding Analysis.

As shown by competition ELISA (FIG. 3A), Hex-hA20 has a 3-fold higheravidity than veltuzumab, suggesting that at least three or more of thesix Fab components are capable of binding simultaneously to the WR2antiidiotype antibody. The binding of Hex-hA20 to CD20 on live cells wascompared with that of veltuzumab by flow cytometry. Hex-hA20 resulted in40% to 50% greater fluorescence intensity than veltuzumab when probed byPE-anti-Fab (FIG. 3B). In contrast, the signal observed for Hex-hA20with PE-anti-Fc was lower than that of veltuzumab. These results areconsistent with Hex-hA20 having four more Fabs than veltuzumab, but onlyone Fc like veltuzumab.

Additional evidence for the higher valency and avidity of Hex-hA20 isprovided by Scatchard analysis of binding to Raji cells (FIG. 3C), whichshowed that the apparent association constant of Hex-hA20 [3.9×10⁸(mol/L)⁻¹] was 4-fold higher (P<0.0001) than that of veltuzumab [1×10⁸(mol/L)⁻¹], whereas an F test determined that the saturation bindingcurves for rituximab and veltuzumab were similar (P=0.1859). Moreimportantly, the number of receptors per cell calculated from themaximum number of binding sites (B_(max)) obtained for Hex-hA20 wasfound to be ¼ of that obtained with veltuzumab (100,000 versus437,5000), suggesting that all six Fabs of Hex-hA20 are capable ofbinding to CD20 on the cell surface, because the same number of CD20molecules would require three times as many veltuzumab to occupy them.The data obtained from the off-rate measurements (FIG. 3D) alsoindicated that Hex-hA20 dissociates 3-fold slower than veltuzumab, whichdissociates 2-fold to 3-fold slower than rituximab (Glennie et al.,2007, Mol Immunol 44: 3823-37).

Antiproliferative Activity.

Based on the MTS assay (FIG. 4A), Hex-hA20 strongly inhibitedproliferation in three Burkitt lymphoma cell lines, Raji, Ramos, andDaudi, with an EC₅₀ of 0.064, 0.15, and 0.15 nmol/L, respectively. Incontrast, veltuzumab in the absence of a cross-linking antibody showeddetectable potency in all three cell lines only at >10 nmol/L. Asexpected, cross-linking of veltuzumab with goat anti-human Fc resultedin significant inhibition of proliferation. For Hex-hA20, cross-linkingdid not increase its potency further in Daudi. Additional experimentscompared the effects of Hex-hA20, Tri-hA20, and Tetra-hA20 on cellproliferation over 5 days by viable cell counting. Representativeresults are shown in FIG. 4B for Raji and Ramos, which show the abilityof Tri-hA20, Tetra-hA20, and Hex-hA20 to prevent 50% or greater cellproliferation without cross-linking at concentrations as low as 0.5nmol/L. Similar results were obtained for Daudi (data not shown). Thecell counting assay also showed that Hex-hA20 (EC₅₀=0.17 nmol/L) wasconsiderably more potent than anti-B 1 (EC₅₀=4.65 nmol/L) when evaluatedwith Ramos for 3 days.

Apoptosis and the Roles of Caspases and Calcium.

Raji cells were treated with the three multivalent anti-CD20 constructsat 0.5 or 5 nmol/L and analyzed with the GUAVA® Nexin assay after 24hours (FIG. 5A, left). Treatment with Tri-hA20, Tetra-hA20, or Hex-hA20resulted in more cells in early apoptosis (12-16%) compared with thebivalent veltuzumab (6-9%) and the untreated control (3%). Comparableresults were obtained with Daudi and Ramos cells (data not shown).

The extent of apoptosis was also assessed for Raji cells treated with 5nmol/L Hex-hA20 or veltuzumab over a 24-hour period using the GUAVA®MultiCaspase assay (FIG. 5A, right). The results at 24 hours were 17%and 6% for Hex-hA20 and veltuzumab, respectively, which agree with thosedetermined by the GUAVA® Nexin assay.

The effect of Z-VAD-FMK (a broad-spectrum caspase inhibitor) onapoptosis induced by Hex-hA20 was examined in Ramos, and the results(not shown) indicated that Z-VAD-FMK at 100 μmol/L completely preventedthe apoptosis induced by antihuman IgM, but not that induced byHex-hA20, suggesting that both caspase-dependent and caspase-independentpathways occur for Hex-hA20. Although CD20 clustering is presumablyachievable either indirectly by cross-linking the antigen-boundveltuzumab with a second antibody or directly via multivalent engagementof Hex-hA20, the former, but not the latter, leads to a rapid rise inintracellular calcium levels (not shown).

Effector Functions.

CDC activity was evaluated in vitro using human complement and Daudicells (FIG. 5B, left). Veltuzumab exhibited potent CDC activity.Surprisingly, Hex-hA20 failed to induce CDC in Daudi cells. BecauseC_(H3)-AD2-IgG-hA20 induces CDC with similar potency as veltuzumab,modification of the carboxyl termini of veltuzumab by the addition ofthe small AD2 peptide does not affect CDC, suggesting that the additionof the four Fab-DDD2 groups apparently prevents complement fixation,despite the ability of Hex-hA20 to bind C1q (data not shown). Hex-hA20and veltuzumab have comparable ADCC (FIG. 5B, right). Thus, the Fc ofHex-hA20 induces ADCC.

Homotypical Adhesion.

Hex-hA20, Tetra-hA20, and Tri-hA20 induced homotypical adhesion of Daudicells, resulting in >50% of cells having medium-size to large-sizeaggregates, whereas under the same conditions, the extent of cellaggregation observed for veltuzumab was similar to that of the untreatedcontrol (not shown).

Membrane Localization of CD20/Hex-hA20.

The distribution of CD20 at the cell surface upon binding to veltuzumabor Hex-hA20 was examined with immunofluorescence microscopy usingcholera toxin subunit B-Alexa Fluor 488 as the reporter for gangliosideGM-1, a common lipid raft marker. Incubation of Daudi cells withveltuzumab or Hex-hA20 led to the formation of membrane patches or capswith punctuate spots, which were superbly matched by the images obtainedwith cholera toxin subunit B, indicating the localization of CD20 inlipid rafts (not shown). Although the fluorescent patterns observed weresimilar, veltuzumab seemed to form larger and fewer patches thanHex-hA20 (not shown).

Serum Stability.

Hex-hA20 was found to have the same stability in serum as veltuzumab,maintaining 86% binding activity after 11 days (not shown). Theseresults are similar to those of the bispecific Tri-Fab complexesreported previously (Rossi et al., 2006, Proc Natl Acad Sci USA103:6841-6).

Pharmacokinetic Analysis.

Using radio-iodinated preparations, it was found that Hex-hA20 cleared4.5 times faster than veltuzumab when given i.v. (not shown), resultingin a 3.7-fold lower mean residence time for Hex-hA20 compared withveltuzumab (127 hours versus 472 hours). When given s.c., Hex-hA20cleared at a much faster rate (T_(1/2) 3 days) than veltuzumab(T_(1/2)>9 days), with both having the same T_(max) of 24 hours (notshown). However, the C_(max) was only half as high for Hex-hA20 comparedwith veltuzumab (12.9 nmol/L versus 25.7 nmol/L).

In Vivo Efficacy.

In the multiple-dose study with the Daudi model, mice were treated withHex-hA20 at 30 μg (q7dx2) and 6 μg (q7dx2). As shown in FIG. 6A, bothtreatments resulted in significantly improved median survival time (MST)when compared with the saline control (66.5 and 42 versus 21 days;P<0.0001). Although no significant difference in survival was observedbetween mice given Hex-hA20 and veltuzumab at the higher doses, itseemed that mice receiving Hex-hA20 at the lower doses had significantlylower MST than those receiving veltuzumab at an equivalent dose(P=0.0044).

We also examined the role of effector cells in inhibiting tumor growthusing the Raji model (FIG. 6B). In those animals depleted of NK cellsand neutrophils, there was no difference between the saline control andmice treated with veltuzumab or Hex-hA20 (MST=17 days for all threegroups). In contrast, nondepleted mice that received Hex-hA20 orveltuzumab had significantly improved survival than the saline control(P=0.0034), with the MST of 59, 41, and 19 days for Hex-hA20,veltuzumab, and untreated, respectively. Importantly, a better treatmentoutcome was observed for Hex-hA20 than for veltuzumab (P=0.05) at thishigher mole-equivalent dose (465 μg Hex-hA20 versus 200 μg veltuzumab).

Discussion

The fact that Tri-hA20, but not veltuzumab, can potently inhibit theproliferation of CD20-positive cells in vitro is consistent with themodel that all three Fabs in Tri-hA20 are capable of simultaneouslybinding to CD20, resulting in clustering of CD20 and the onset of signaltransduction, which leads us to conclude that a minimum valency of 3 isrequired for an anti-CD20 antibody to effectively induce growthinhibition without cross-linking.

Based on their efficacy in certain in vitro assays, anti-CD20 mAbs havebeen classified by Cragg and colleagues (Cragg et al., 2003, Blood101:1045-52) as either type I, represented by rituximab, or type II,represented by tositumomab. We note that Hex-hA20 exhibits biologicalproperties attributable to both type II (for example, negative for CDCand calcium mobilization; positive for antiproliferation, apoptosis, andhomotypical adhesion) and type I (for example, positive for traffickingto lipid rafts). Thus, one effective approach to converting a type Ianti-CD20 mAb to a type II can be achieved by making the type I mAbmultivalent. Preliminary investigation of the signaling pathwayindicates that Hex-hA20 induces caspase-dependent, as well ascaspase-independent, apoptosis. Additional studies are in progress toidentify the subcellular events associated with the binding of CD20 byHex-hA20 or Tri-hA20, which may reveal unequivocally the molecularfactors that account for the antiproliferative potency of a multivalentanti-CD20 antibody with defined composition.

These results demonstrate that production of multivalent antibodies bythe DNL methodology produces superior efficacy. The skilled artisan willrealize that multivalent DNL complexes comprising one or more anti-HIVantibodies or fragments thereof, such as P4/D10, 2G12, 2F5 or 4E10, maybe constructed using the same technique.

Example 6 PEGylated DNL Complexes

In certain embodiments, PEG moieties may be incorporated into DNLcomplexes, for example to provide for a reproducible and homogeneousPEGylated product of an effector moiety. As a first step, the followingpeptide subunits capable of covalent conjugation to PEG moieties forincorporation into DNL complexes were synthesized on a commercialpeptide synthesizer. Fmoc-Cys(t-Buthio)-OH was used to add the SS-tburesidue. Fmoc-Gly-EDANS resin was used to attach the G-EDANS moiety.

IMP350 (SEQ ID NO: 93) CGQIEYLAKQIVDNAIQQAGC(SS-tbu)-NH₂ IMP360(SEQ ID NO: 94) CGQIEYLAKQIVDNAIQQAGC(SS-tbu)-G-EDANS IMP421(SEQ ID NO: 95) Ac-C-PEG₃-C(S-tBu)GQIEYLAKQIVDNAIQQAGC(S-tBu)G-NH₂

Generation of IMP362, IMP413 and IMP457

The two linear PEG-AD2 modules were prepared by coupling IMP360 tomPEG-OPTE (Nectar Therapeutics, San Carlos, Calif.) of 20-kDa or 30-kDa,resulting in IMP362 or IMP413, respectively. To prepare IMP362, IMP360(11.5 mg) was mixed with 20-kDa mPEG-OPTE (127 mg) in 7 mL of 1 MTris-HCL, pH 7.8. Acetonitrile (1 mL) was added to dissolve somesuspended material. The reaction was stirred at room temperature for 4 hto effect the attachment of mPEG to the amino-terminal cysteine via anamide bond. Subsequently, 41 mg of Tris [2-carboxyethyl]phosphinehydrochloride (TCEP) and 43 mg of cysteine were added to de-protect theremaining cysteine. The reaction mixtures were stirred for 1 h anddesalted using PD-10 columns, which had been equilibrated with 20%methanol in water. The samples were lyophilized to obtain approximately150 mg of IMP362. IMP413 was made similarly using 30-kDa mPEG-OPTE (190mg). IMP457 was made similarly using mPEG2-MAL-40K (Nectar Therapeutics)to obtain the branched PEG-AD2 module (IMP457).

Construction of IFN-α2b-DDD2-pdHL2 for Expression in Mammalian Cells

The cDNA sequence for IFN-α2b was amplified by PCR using a full lengthhuman IFNα2b cDNA clone (Invitrogen ULTIMATE™ ORF human clone cat#HORF01Clone ID IOH35221) as a template and the followingoligonucleotides as primers:

IFNA2 Xba I Left (SEQ ID NO: 96)5′-TCTAGACACAGGACCTCATCATGGCCTTGACCTTTGCTTTACTGG-3′ IFNA2 BamHI right(SEQ ID NO: 97)5′-GGATCCATGATGGTGATGATGGTGTGACTTTTCCTTACTTCTTAAACTTTCTTGC-3′

The resulting secreted protein consists of IFN-α2b fused at itsC-terminus to a polypeptide consisting of SEQ ID NO:98.

(SEQ ID NO: 98) KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

The PCR amplimer was cloned into the pGEM®-T vector. A DDD2-pdHL2mammalian expression vector was prepared for ligation with IFN-α2b bydigestion with XbaI and Bam HI restriction endonucleases. The IFN-α2bamplimer was excised from PGEMT® with XbaI and Bam HI and ligated intothe DDD2-pdHL2 vector to generate the expression vectorIFN-α2b-DDD2-pdHL2.

IFN-α2b-DDD2-pdHL2 was linearized by digestion with Sail enzyme andstably transfected into Sp/EEE myeloma cells by electroporation forproducing the expressed protein (see. e. g., U.S. Pat. No. 7,537,930,the Examples section of which is incorporated herein by reference).

Preparation and Purification of α2b-362 (IFN-α2b-DDD2-IMP362)

The structure of α2b-362 has two copies of IFNα2b-DDD2 coupled to a 20kDa PEG-AD. A DNL reaction was performed by the addition of 11 mg ofreduced and lyophilized IMP362 in 10-fold molar excess to 2.25 mg (3.5ml) of IFN-α2b-DDD2 in 250 mM imidazole, 0.02% Tween 20, 150 mM NaCl, 1mM EDTA, 50 mM NaH₂PO₄, pH 7.5. After 6 h at room temperature in thedark, the reaction mixture was dialyzed and purified by columnchromatography on a cation-exchange resin.

The DNL reaction resulted in the site-specific and covalent conjugationof IMP362 with a dimer of IFN-α2b. Overall, the DNL reaction resulted ina near quantitative yield of a homogeneous product that was >90% pureafter purification by cation-exchange chromatography (not shown).

The PEGylated products α2b-457 (IFN-α2b-DDD2-IMP457) and α2b-413(IFN-α2b-DDD2-IMP413) were prepared by similar techniques.

Pharmacokinetics

The study was performed in adult female Swiss-Webster mice (˜35 g). Eachreagent (test and control) was administered at equimolar protein doses(3 μg of rhuIFN-α2a, 5 μg of PEGINTRON®, 11 μg of α2b-362, and 13 μg ofα2b-413) as a single bolus i.v. injection. Mice were bled via theretro-orbital method at various time-points (pre-dose, 5-min, 2-, 8-,24-, 48-, 72-, 96-, and 168-h post-injection). The blood was allowed toclot, centrifuged, and the serum was isolated and stored at −70° C.until assayed for IFN-α concentration and subsequent PK-analysis.

The PK properties of each agent are summarized in Table 6. As expected,rhIFN-α2a had the most rapid clearance from the blood of injected mice.Its clearance was approximately 3-fold faster than the PEGINTRON® andmore than 13-fold faster than the DNL-IFN reagents. The PEGINTRON® wasin turn cleared greater than 4-fold faster than α2b-362 or α2b-413.There was little difference in the elimination rates between α2b-362 andα2b-413. In terms of mean residence time (MRT), there is a clearcorrelation with size among the various reagents. The 19-kDa rhIFN-α2ahad a MRT that was 7-fold less than the 31 kDa PEGINTRON® (0.7 h versus5.1 h, respectively), which had a 2-fold lower MRT when compared to the70 kDa α2b-362 (10.3 h). The MRT for the 80 kDa α2b-413 (21.7 h) was 2-

TABLE 6 Blood Pharmacokinetic Analysis of Interferon-α2b Containing DNLMolecules Administered as Intravenous Injections to Naïve Swiss-WebsterMice. IFN Elimination Animal Dose C_(max) T_(1/2α) T_(1/2β) AUC_(0.08)Rate MRT_(0.08) Number (pmol) (pM) (hours) (hours) (h * pM) (1/h) (h)Recombinant Human Interferon-α2a Animal No. 1 160 16,411 0.29 10.537,011 2.34 0.63 Animal No. 2 160 21,835 0.31 7.14 10,147 2.15 0.78 Mean160 19,123 0.30 8.84 8,579 2.25 0.71 PEGINTRON ® Animal No. 1 160 87,0900.53 6.29 137,790 0.63 5.42 Animal No. 2 160 105,774 0.43 5.11 150,9050.70 4.79 Mean 160 96,432 0.48 5.70 144,348 0.67 5.11 IFN-α2b-IMP362Animal No. 1 320 60,833 1.72 7.54 379,462 0.16 9.03 Animal No. 2 32097,089 1.43 10.14 570,336 0.17 11.56 Mean 320 78,961 1.58 8.84 474,8990.17 10.30 IFN-α2b-IMP413 Animal No. 1 320 152,923 0.69 12.85 1,012,4700.15 16.75 Animal No. 2 320 100,495 4.03 28.53 1,179,056 0.09 26.56 Mean320 126,709 2.36 20.69 1,095,763 0.12 21.66fold longer than α2b-362. Finally, a test for bioequivalence showed thatnone of the reagents tested were the same in terms of PK, indicatingthat the differences are genuine (i.e., circulating half-life forα2b-413>α2b-362>PEGINTRON®>rhIFN-α2a).

In Vivo Efficacy

An in vivo tumor therapy study demonstrated that the DNL-PEGylatedinterferons were more potent and longer-lasting compared to PEGINTRON®.Eight-week-old female C.B.-17 SCID mice were injected i.v. with a humanBurkitt's lymphoma cell-line (Daudi) at 1.5×10⁷ cells per animal.Equivalent units of activity of PEGINTRON®, α2b-362 and α2b-413 wereadministered once every 7 days via s.c. injection in either the left orright flank at three different doses (3500, 7000, and 14000 Units).Therapy commenced 1 day after the Daudi cells were transplanted.

Survival curves were generated. PEGINTRON®, α2b-362, and α2b-413 alldemonstrated significant improvement in survival when compared to salinecontrol mice (P<0.0016) (not shown). Except for the 3,500 IU dose ofα2b-362, both α2b-413 and α2b-362 were superior to PEGINTRON® whenadministered at equal activity doses (P<0.0027) (not shown). α2b-362showed more than twice the potency of PEGINTRON® (not shown). Doses of7,000 IU and 3,500 IU of α2b-362 were superior to 14,000 IU (P=0.0016)and 7,000 IU (P=0.0027) doses of PEGINTRON®, respectively (not shown).α2b-413 is more than four times as potent as PEGINTRON® since a 3,500 IUdose of the former was superior to 14,000 IU of the latter (P=0.0027)(not shown). α2b-413 was significantly better than α2b-362 (P<0.0025)when administered at equivalent doses. However, there were nostatistically significant differences among the three doses of α2b-413,even though the 14,000 IU dose resulted in a median survival of 60 daysin comparison to the 3,500 IU dose and its 46 day median survival(P=0.1255). The in vivo efficacy observed for α2b-362, α2b-413, andPEGINTRON® thus correlate well with the PK data.

The increased bioavailability of α2b-362 and α2b-413 demonstrated by PKanalysis contributes to the enhanced in vivo anti-tumor potency ofDNL-PEGylated IFNα. In turn, these two factors allow for a less frequentdosing schedule used in tumor therapy. This was demonstrated with asimilar in vivo tumor therapy study as above, in which equal units ofactivity of PEGINTRON® or α2b-413 were administered with varied dosingschedules. Each reagent (test and control) was administered at 14,000 IUvia a s.c. injection in either the left or right flank.

All the IFN-IMP413-treated mice had significantly improved survival whencompared to those animals treated at the same schedule with PEGINTRON®(P<0.0097) (not shown). Of note, those mice treated every other weekwith IFN-IMP413 (q2wk×4) not only had significantly improved survival incomparison to those treated with PEGINTRON® at the same schedule(MST=>54 days versus 28 days, respectively; P=0.0002), but were alsosignificantly better than those animals treated weekly (q7dx4) withPEGINTRON® (MST=36.5 days; P=0.0049) (not shown). Further, survival ofmice treated every three weeks with IFN-IMP413 (q3wk×4) wassignificantly better than those treated with PEGINTRON® every two weeks(MST=54 days versus 28 days; P=0.002) and approaches significance whencompared to those treated weekly with PEGINTRON® (P=0.0598) (not shown).

In another study, we found that administering α2b-413 at 14,000 IU every4 weeks increased the median survival to 56 days from 23 days of thesaline control and was more potent than PEGINTRON® given 14,000 IU everyweek (not shown).

For a better comparison with PEGASYS®, we conjugated IFNα2b-DDD2 toIMP457, an AD2-module of 40-kDa branched PEG, and obtained a resultingα2b-457. The in vitro biological activities of α2b-457 were determinedby three different assays to be lower than PEGINTRON®, comparable toα2b-413, and considerably higher than PEGASYS® (not shown). The PK dataobtained in mice with a single s.c. injection indicate a longercirculating half-life of α2b-457 than either α2b-413 or PEGASYS®, withall three clearing much slower than PEGINTRON® (not shown).

When given once every four weeks at a low dose of 20 μmol, α2b-457 wasmore effective than PEGINTRON® given as a mole-equivalent dose onceweekly. Administration of α2b-457 extended the median survival ofDaudi-bearing mice to 47 days from 23 days when compared to the salinegroup (not shown). In the same study, α2b-457 at 20 pmol wassignificantly better than either α2b-413 or PEGINTRON® at 20 pmol(MST=47 days versus 41 and 37 days, respectively; P<0.0151) (not shown).The 20 pmol dose of α2b-413 also improved survival in comparison toPEGINTRON® (P=0.002) (not shown). At 10 pmol, there was no differencebetween α2b-457 and α2b-413 but both significantly improved survivalover PEGINTRON® treated mice (P<0.001) (not shown).

These studies demonstrated that PEGylation of therapeutic agents usingthe DNL technique resulted in improved and long-lasting efficacy, evenwhen compared with other PEGylated forms of IFNα2b, allowing for lessfrequent dosing. The skilled artisan will realize that DNL PEGconjugates may provide similarly improved pharmacokinetics and/orefficacy for anti-HIV therapeutic agents.

Example 7 Anti-HIV DNL Complex

Among the various antibodies that neutralize HIV-1, the murineanti-gp120 antibody, P4/D10, is distinguished by its ability to induceantibody-dependent cell-mediated cytotoxicity (ADCC) to eliminateinfected T cells that express the antigenic gp120 epitope bound byP4/D10 (Broliden et al., 1990, J Virol 64:936-40). Enhanced potency wasalso shown in Example 1 above for doxorubicin-conjugated P4/D10 toneutralize and inhibit intercellular spread of HIV infection in vitro,as well as to protect against HIV-1/MuLV infection in vivo (Johansson etal., 2006, AIDS 20:1911-15).

The Dock-and-Lock (DNL) method was used to generate a DNL complexcomprising P4/D10 IgG, or other antibodies or fragments thereof, alongwith one or more anti-HIV agents.

In a preferred embodiment illustrated herein, the anti-HIV agent was theT20 HIV fusion inhibitor (enfuvirtide, FUZEON®) (Asboe, 2004, HIV ClinTrials 5:1-6). However, the skilled artisan will realize that otheranti-HIV therapeutic agents known in the art, described in more detailabove, may be utilized either attached to an anti-HIV DNL complex orseparately administered before, simultaneously with, or after ananti-HIV DNL complex.

The primary target HIV patient population for the subject DNL complexesis individuals failing HAART therapy, where several doses of the DNLconjugates may effectively reduce the number of infected cells andcirculating virions. A secondary patient population is individuals oneffective HAART, with the goal to reach and delete the few persisting,virus-producing cells.

In a preferred embodiment illustrated in FIG. 7, the DNL method was usedto develop a novel class of anti-HIV agents that comprise multiplecopies of enfuvirtide (T20) linked to a chimeric, human or humanizedantibody with specificity for HIV-1. The C-terminal end of each heavychain of an IgG antibody was attached via a short linker to an AD2moiety (SEQ ID NO:4) and expressed as a fusion protein as described inthe Examples above. The T20 HIV fusion inhibitor was attached to a DDD2moiety (SEQ ID NO:2) and also expressed as a fusion protein. Two copiesof the DDD2 moiety spontaneously form a dimer that binds to the AD2moiety, forming a DNL complex comprising one IgG antibody and fourcopies of T20. The preclinical results obtained to date indicate thatsuch IgG-(T20)₄ conjugates should allow less frequent dosing than withunconjugated T20 to block entry of HIV-1 into T cells, neutralizecell-free HIV-1 and eliminate HIV-infected cells.

The DDD2-T20 amino acid sequence is shown below in SEQ ID NO:99. Thesequence of DDD2 is underlined. This is followed by a short linker andhinge region and a polyhistidine tag for affinity purification. Thesequence of T20 at the C-terminal end is in bold. DDD2-T20 was producedin E. coli, shown by LC-MS to have the exact mass predicted from thedesigned amino acid sequence (data not shown), and was used to make DNLcomplexes, as described below.

DDD2-T20 (SEQ ID NO: 99)MCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARAEFPKPSTPPGSSGHHHHHHGSYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

P4/D10 is a murine antibody that may induce human anti-mouse antibodies(HAMA) when administered to human subjects. Chimeric or humanized formsof P4/D10 would be more suitable for human therapeutic use. A chimericP4/D10 (cP4/D10) was constructed by grafting the V_(H) and V_(K)sequences of P4/D10 (FIG. 8) onto the constant region sequences of ahuman IgG1. The resulting cP4/D10 has the same DNA and amino acidsequences as P4/D10 in the variable domains (FIG. 9). cP4/D10 wasprepared and its binding affinity for gp160 (comprising both gp120 andgp41) was found to be comparable to that of murine P4/D10 (FIG. 10A).The binding affinity of cP4/D10 for the reactive epitope of P4/D10located in the V3 loop of gp120 was also found to be comparable withthat of P4/D10 and was not affected by the presence of 8 M urea (FIG.10B).

Besides cP4/D10, which is a representative of neutralizing anti-HIV mAb,the potential of conjugating T20 to other antibodies to enhance thepotency of T20 was investigated and demonstrated in vitro with twohumanized IgG1 antibodies, h734 (anti-indium-DTPA) and hLL2(epratuzumab; anti-CD22), as well as the Fab of hA20 (veltuzumab,anti-CD20). The results (FIG. 11 to FIG. 13) show that the efficacy ofan HIV-fusion inhibitor in general, and T20 in particular, may beimproved by incorporating it into a DNL complex with a wide variety ofantibodies or antibody fragments that are neither neutralizing nordirected against the cell-surface receptor (CD4) or coreceptors (CCR5and CXCR4) of HIV. A further increase in efficacy may be achieved bycoadministering a T20-containing DNL complex with a broadly neutralizingantibody such as 2G12 (Hessell et al., 2009, PLoS Pathogens 5:e1000433)and/or an antibody targeting CD4, CCR5 or CXCR4. Table 7 below listsselective T20-containing DNL conjugates made to date and theirrespective antibody component. SE-HPLC analysis of hLL2-(T20)₄,cP4/D10-(T20)₄, and hA20-Fab-(T20)₂ along with their corresponding AD2modules was consistent with the proposed structures of the DNLcomplexes.

TABLE 7 DNL-T20 complexes. Advantages over DNL Complex Antigen AntibodyUnconjugated T20 h734-(T20)₄ Indium- h734 Improved PK and DTPA potentialADCVI hLL2-(T20)₄ CD22 hLL2 Improved PK and potential ADCVI cP4/D10-gp120 hP4/D10 Improved PK, (T20)₄ (V3 loop) neutralizing HIV, killinginfected T cells hA20-Fab- CD20 hA20 Increased efficacy (T20)₂

DDD2-T20 was produced in E. coli and used with the h734-IgG-AD2 andhLL2-IgG-AD2 modules to prepare h734-(T20)₄ and hLL2-(T20)₄, two DNLcomplexes that are not specific for HIV. FIG. 11A compares the in vitroneutralization activities of DDD2-T20 h734-(T20)₄, and the unconjugatedT20 (FUZEON®) by p24 ELISA (Johansson et al., 2006, AIDS 20:1911-15),showing approximately equal efficacy when the concentration of eachagent in μg/ml is used for the X-axis. Because the molecular weight ofh734-(T20)₄ is significantly higher than either DDD2-T20 or T20, thesedata were replotted in molar concentrations (FIG. 11B). When plotted ona molar basis, the superior potency of h734-(T20)₄ over both DDD2-T20and T20 is shown (FIG. 11B).

A comparison of the potency of h734-(T20)₄ with the published data ofselective HIV fusion inhibitors is provided in Table 8. The h734-(T20)₄complex exhibited an EC₅₀ of about 0.1 nM, compared to 1 to 2 nM forT20. The h734-(T20)₄ complex exhibited an EC₉₀ of about 0.6 nM, comparedto about 10 nM for T20. The values for EC₅₀ and EC₉₀ of the h734-(T20)₄DNL complex were lower than those reported for any other HIV fusioninhibitor (Table 8). These results show that even when a non-targetingantibody was used, its DNL complex with T20 exhibited a substantiallyhigher efficacy than unconjugated T20, which is a notable advantage overconjugates of fusion inhibitors made with other techniques than DNL. Forexample, EP40111, a PEG-pentasaccharide conjugate of T20, had asubstantially decreased potency compared to unconjugated T20. PC-1505, ahuman serum albumin conjugate of C34 (a T20 analog that shows somewhatimproved efficacy, but similar half-life compared to T20), did not showan improved potency over T20.

TABLE 8 Relative Potencies of HIV Fusion Inhibitors Anti-HIV potencyHIV-fusion inhibitor # T20 or C34 EC50 (nM) EC90 (nM) T20 1 1 to 2 ~10EP40111 1 (T20) 100 NA Fc-T20 2 5 NA 734-T20 4 ~0.1 ~0.6 C34 1 0.6 2.8HSA-C34 (PC-1505) 1 1.8 13.5

The in vitro efficacy of the DNL agents is further demonstrated in FIG.12A-D, which compares the potencies of P4/D10, cP4/D10, cP4/D10-(T20)₄,h734-(T20)₄, and hLL2-(T20)₄ for neutralization of HIV-1_(IIIB) inJurkat T cells and HIV-1₆₇₉₄ in PBMCs. These results indicate thefollowing. cP4/D10 and P4/D10 were equivalent in their potencies toneutralize HIV-1_(IIIB) in Jurkat T cells (FIG. 12A). cP4/D10-(T20)₄ wasmore potent than cP4/D10 in neutralizing both HIV-_(1IIIB) and HIV-1₆₇₉₄(FIG. 12B-D). Both hLL2-(T20)₄ and h734-(T20)₄ were surprisingly morepotent than cP4/D10-(T20)₄ in neutralizing HIV-1 (FIG. 12D). Theunconjugated hLL2 and hMN-14 IgG had no neutralization activity (FIG.12A-D). Table 9 summarizes the EC50 values estimated from the resultsshown in FIG. 12A-@.

TABLE 9 Relative Potencies of Unconjugated T20, Unconjugated Antibodiesand Conjugated DNL Complexes. HIV-1_(IIIB) HIV-I₆₇₉₄ # of Jurkat T PBMCsMW (Da) T20 50TCID₅₀ 100TCID₅₀ 50TCID₅₀ 100TCID₅₀ T20 4,492 1 ~9 ~9 ~9P4/D10 ~150,000 0 1 2 1 8 cP4/D10 ~150,000 0 2 cP4/D10-(T20)₄ ~200,000 4<0.4 <0.4 1.6 h734-(T20)₄ ~200,000 4 0.2 hLL2-(T20)₄ ~200,000 4 <0.2 0.1<0.2 <0.2

The potential use of DNL-T20 complexes for treating latently infectedcells was investigated by measuring the neutralizing activity ofhLL2-(T20)₄ against HIV-_(1IIIB) in PBMCs over a period of 30 daysfollowing activation by SAHA (suberoylanilide hydroxamic acid) (FIG.13). For comparison, cP4/D10, T20 and hLL2 were also included in thestudy. The results obtained indicate that a substantial and persistentincrease in HIV replication, measured by either p24 ELISA (FIG. 13A) orp24-positive cultures (FIG. 13B), was observed in SAHA-added mediumthroughout the 30-day period, which could be nearly completelysuppressed by hLL2-(T20)₄, cP4/D10 or T20. On day 30, as shown in FIG.13C, each of the three agents reduced p24-positive cultures to less than5% of the medium+SAHA control. Surprisingly, hLL2 also reducedp24-positive cultures to about 50% of the medium+SAHA control. hLL2binds to the CD22 antigen, which is present on the surface of mature Bcells.

The in vivo stability of hLL2-(T20)₄ was determined as follows. NaïveSCID mice (11 total) were injected s.c. with hLL2-(T20)₄ (100 μg; 500pmol). Serum samples were collected at 0.5, 6, 24 and 72 h from 2, 3, 3,and 3 mice, respectively, and stored at −70° C. until analysis by ELISA.A parallel study was performed with hLL2 IgG (75 μg; 500 pmol) in thesame fashion. The serum samples from mice injected with hLL2-(T20)₄ wereexamined by two different ELISAs, one designed to quantify only theintact hLL2-(T20)₄ and the other to quantify all hLL2-containingspecies, with or without the linked T20. For quantification ofhLL2-(T20)₄, plates were coated with F(ab′)₂-specific, goat anti-humanIgG, and the captured antibodies probed with a mouse anti-DDD2 mAb (5E3)developed in house, followed by HRP-conjugated goat-anti-mouse. Formeasuring all hLL2-containing species, plates were coated withanti-human F(ab′)2 and the captured antibodies were probed with a ratanti-id mAb to hLL2 (WN), followed by HRP-conjugated goat anti-ratantibodies. The second assay was also used for measuring the serumlevels of hLL2. The results shown in FIG. 14 indicate that hLL2-(T20)₄appears to be stable in vivo at least for 3 days, since the serumconcentrations measured by the two assays were comparable at 6, 24 and72 h. The bioavailability of hLL2-(T20)₄ at 72 h was about half thebioavailability of hLL2.

Antibodies with a broad and effective HIV neutralization activity arecontinuously being identified or engineered (Burton and Weiss, Science2010; 329:770-3) and some of them may have superior properties for usein DNL constructs. Additional antibodies, such as anti-CD4, andalternative HIV-inhibitors, such as next-generation fusion inhibitors,may also be used as components of the DNL conjugates. Enhanced efficacymay also be achieved with the co-administration of unconjugatedantibodies that are themselves effective in monotherapy of HIV.

Humanized and fully human mAbs targeting multiple glyco-epitopes of HIVcarbohydrates and glycoproteins will effectively target HIV-infectedcells and virions during passive immunization against early HIV-1infection or HIV-1 during effective or failing HAART. The HIV-specifictargeting would be further aided by a molecule inserting itself at thetransmembrane region of HIV virions and/or infected cells. As a result,the DNL conjugates of the present design should more selectively targetthe infected cells than non-infected cells.

The skilled artisan will realize that other antibodies and/or HIVtherapeutic agents may be incorporated into DNL constructs using thetechniques described above. Examples of other HIV therapeutic agentsinclude, but are not limited to, sCD4-D1-D2 (West et al., 2010, J Virol.84:261-69), CP32M (He et al., PNAS 2008; 105:16332-7), IZN17 (Eckert andKim, PNAS 2001; 98:11187-92), C34 (Stoddart et al., J Biol Chem 2008;283:34045-52), T1144 (Dwyer et al., PNAS 2007; 104:12772-7), C52L (Denget al., Biochemistry 2007; 46:4360-9), CCR5 antagonists such asmaraviroc or vicriviroc; and agents such as abacavir, amdoxovir,AOP-RANTES, apricitabine, atazanavir, bevirimat, BMS-378806, calanolideA, CCR5, CD4, ceragenin, cobicistat, cyanovirin-N, darunavir,diarylpyrimidines, didanosine, dolutegravir, efavirenz, elvitegravir,elvucitabine, emtricitabine, epigallotachen gallate, festinavir,fosamprenavir, foscarnet, griffithsin, globoidnan A, hydroxycarbamide,indinavir, KP-146, lamivudine, lefinavir, lersivirine, lopinavir,miltefosine, MK-2048, nelfinavir, nevirapine, racivir, raltegravir,ritonavir, saquinavir, selicicib, stafudine, stampidine, stavudine, T61,T651, T1249, T2635, Tat antagonists, tenofovir, tipranavir,trichosanthin, TRIM5alpha, vivecon, zalcitabine, zidovudine orzidovudine. Such other anti-HIV therapeutic agents may be attached orincorporated into the DNL complex, or may alternatively beco-administered to the subject before, concurrently with or after theDNL complex.

Other antibodies of potential use include anti-CD4 antibodies such asibalizumab (Bruno and Jacobson, 2010, J Antimicrob Chemother65:1839-41), anti-Leu3a, L120, OKT4A, 13B8.2 or L71; anti-CCR5antibodies such as NBP1-43335, ab10397, 2D7, HGS004, MC-1, MC-4, MC-5,PA9, PA14 or PRO140 (see, e.g., Lopalco, 2011, J Transl Med 9:S4); orneutralizing anti-HIV antibodies such as 2G12 (Armbruster et al., J.Antimicrob. Chemother. 54:915-20, 2004), 2F5 (Bryson et al., Protein andPeptide Letters, 8:413-18, 2001), 3D6 (Ruker et al., Ann. NY Acad. Sci.646:212-19, 1991), b12 (e.g., Wu et al., J Virol 2006, 80:2585), X5(Moulard et al., Proc Natl Acad Sci 2002, 99:6913-18), C37 (Cao et al.,DNA and Cell Biology, 12:836-41, 2004), 1ACY, 1F58, 1GGGC (Berry et al.,Proteins, 45:281-82, 2001) or 4E10 (Cardoso et al., 2005, Immunity22:163-73). The skilled artisan will realize that DNL complexescomprising any antibody or antigen-binding fragment thereof may beincorporated into a DNL complex using the methods described herein.

In alternative embodiments, HIV therapeutic agents such as T20 areincorporated into DNL constructs with PEG, as described in Example 6above, to provide improved pharmacokinetic properties and decreasedfrequency of administration.

Example 8 PEGylated Anti-HIV Agent DNL Complex

A PEG-AD2 moiety is prepared as described in Example 6 above, selectedfrom IMP362, IMP413 and 1 MP457. T20-DDD2 is prepared as described inExample 7 above. A DNL complex is formed from the PEG-AD2 and T20-DDD2,comprising one PEG moiety attached to two T20 moieties. The PEGylatedT20 DNL complex shows comparable efficacy and over an order of magnitudehigher serum half-life than unconjugated T20, allowing weekly instead ofdaily administration. A decreased incidence of injection site adversereactions is observed with the DNL complex compared to unconjugated T20.

Example 9 DNL Complex with Humanized Anti-HIV Antibody

Chimeric P4/D10 antibody prepared as described in Example 7 above isused to prepare a humanized P4/D10 (hP4/D10), according to Leung et al.(1995, Mol. Immunol., 32: 1413), by attaching the murine CDR sequencesto human antibody framework region (FR) and constant region sequences.The human antibody FR sequences are constructed using the same human IgGdonor FRs as the humanized anti-CD22 antibody epratuzumab (Leung et al.,Mol Immunol 1995; 32: 1413-1427). Specifically, FR1, FR2, and FR3 of thehuman EU antibody and FR4 of the human NEWM antibody are selected forthe heavy chain and the FRs of the human REI antibody are selected forthe light chain of the hP4/D10 antibody. As disclosed in U.S. Pat. No.7,151,164, key murine residues are retained in the FRs to maintain thebinding specificity and affinity of hP4/D10 for gp120.

The Vκ sequence for the MAb is amplified using the primers VK1BACK andVK1FOR (Orlandi et al, 1989). The V_(H) sequence is amplified using theprimer pair VH1BACK/VH1FOR (Orlandi et al., 1989). PCR reaction mixturescontain 10 μl of the first strand cDNA product, 10 μl of 10×PCR buffer[500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl₂, and 0.01% (w/v)gelatin] (Perkin Elmer Cetus, Norwalk, Conn.), 250 μM of each dNTP, 200nM of the primers, and 5 units of Taq DNA polymerase (Perkin ElmerCetus) are subjected to 30 cycles of PCR. Each PCR cycle consists ofdenaturation at 94° C. for 1 min, annealing at 50° C. for 1.5 min, andpolymerization at 72° C. for 1.5 min. Amplified Vκ and V_(H) fragmentsare purified on 2% agarose (BioRad, Richmond, Calif.). The humanized Vgenes are constructed by a combination of long oligonucleotide templatesyntheses and PCR amplification as described by Leung et al. (Mol.Immunol., 32:1413 (1995)).

PCR products for Vκ are subcloned into a pBR327-based staging vector,VKpBR, that contains an Ig promoter, a signal peptide sequence andconvenient restriction sites to facilitate in-frame ligation of the VκPCR products. PCR products for V_(H) are subcloned into thepBluescript-based VHpBS. Individual clones containing the respective PCRproducts are sequenced by the method of Sanger et al. (Proc. Natl. Acad.Sci., USA, 74: 5463 (1977)).

Expression cassettes containing the Vκ and V_(H) sequences, togetherwith the promoter and signal peptide sequences, are excised from VKpBRand VHpBS, respectively, by double restriction digestion asHindIII-BamHI fragments. The Vκ and VH expression cassettes areassembled in the modified staging vectors, VKpBR2 and VHpBS2, excised asXbaI/BamHI and XhoI/BamHI fragments, respectively, and subcloned into asingle expression vector, pdHL2, as described by Gilles et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)). The expression vector is transfected into Sp-EEE,Sp-ESF or Sp-ESF-X mammalian host cells for expression and antibodyproduction.

Antibodies are isolated from cell culture media as follows. Cells aregrown as a 500 ml culture in roller bottles using HSFM. Cultures arecentrifuged and the supernatant filtered through a 0.2 μl membrane. Thefiltered medium is passed through a protein A column. The resin is thenwashed with about 10 column volumes of PBS and protein A-bound antibodyis eluted from the column with 0.1 M glycine buffer (pH 3.5) containing10 mM EDTA. Peak fractions are pooled, dialyzed against PBS, and theantibody concentrated with a CENTRICON® 30 concentrator (Amicon,Beverly, Mass.).

Purified hP4/D10 is attached to AD2 moieties as described in Example 7above. The CP32M fusion inhibitor peptide is attached to DDD2 andexpressed as a fusion protein as described in Example 7 above. A DNLcomplex comprising hP4/D10-AD2 attached to DDD2-CP32M is prepared asdescribed for the 734-T20 DNL complex in Example 7 above. ThehP4/D10-CP32M DNL complex shows significantly improved efficacy andequivalent serum half-life, compared to the 734-T20 DNL complex.

Example 10 Use of Other Anti-HIV Antibodies for DNL Complex Formation

The 2G12 anti-HIV antibody is purchased from Polymun Scientific (Vienna,Austria). An AD2-2G12 fusion protein is prepared as described in Example7 above. DDD2-T20 is prepared as described in Example 7 above. A DNLcomplex comprising 2G12-AD2 attached to DDD2-T20 is prepared asdescribed for the 734-T20 DNL complex in Example 7 above. The 2G12-T20DNL complex shows significantly improved efficacy and equivalent serumhalf-life, compared to the 734-T20 DNL complex.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the methods described hereinwithout departing from the concept, spirit and scope of the invention.More specifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A method of treating an HIV infection comprisingadministering to a subject a DNL complex comprising: a) an antibody orantigen-binding fragment thereof that binds to an antigen selected fromthe group consisting of gp120, gp41, CD4 and CCR5, conjugated to aprotein kinase A protein (AKAP) anchoring domain (AD) moiety; and b) atleast one anti-HIV therapeutic agent conjugated to a human proteinkinase A (PKA) regulatory subunit dimerization and docking domain (DDD)moiety; wherein two copies of the DDD moiety bind to one copy of the ADmoiety to form the DNL complex.
 2. The DNL complex according to claim 1,wherein the antibody or fragment thereof and AD moiety comprise a firstfusion protein and the anti-HIV therapeutic agent and DDD moietycomprise a second fusion protein.
 3. The method according to claim 1,wherein the antibody is selected from the group consisting ofibalizumab, anti-Leu3a, L120, OKT4A, 13B8.2, L71, NBP1-43335, ab10397,2D7, HGS004, MC-1, MC-4, MC-5, PA9, PA14, PRO140, 2G12, 2F5, 3D6, b12,C37, E4D10, 1ACY, 1F58, 1GGGC, VRC01, HJ16, 17b and P4/D10.
 4. Themethod according to claim 1, wherein the antibody is a P4/D10 antibody,a chimeric P4/D10 antibody or a humanized P4/D10 antibody.
 5. The methodaccording to claim 1, wherein the antibody is a human or humanizedantibody that binds to the same epitope of gp120 as a P4/D10 antibody.6. The method according to claim 1, wherein the anti-HIV therapeuticagent is selected from the group consisting of T20, T61, T651, T1249,T2635, CP32M, IZN17, C34, T1144, C52L, CCR5 antagonists, maraviroc,vicriviroc, abacavir, amdoxovir, AOP-RANTES, apricitabine, atazanavir,bevirimat, BMS-378806, calanolide A, CCR5, CD4, ceragenin, cobicistat,cyanovirin-N, darunavir, diarylpyrimidines, didanosine, dolutegravir,efavirenz, elvitegravir, elvucitabine, emtricitabine, epigallotachengallate, festinavir, fosamprenavir, foscarnet, griffithsin, globoidnanA, hydroxycarbamide, indinavir, KP-146, lamivudine, lefinavir,lersivirine, lopinavir, miltefosine, MK-2048, nelfinavir, nevirapine,racivir, raltegravir, ritonavir, saquinavir, sCD4-D1-D2, selicicib,stavudine, stampidine, Tat antagonists, tenofovir, tipranavir,trichosanthin, TRIM5alpha, vivecon, zalcitabine and zidovudine.
 7. Themethod according to claim 1, wherein the anti-HIV therapeutic agent isnot a protein or peptide and is covalently attached to DDD2.
 8. Themethod according to claim 1, wherein the anti-HIV therapeutic agent isselected from the group consisting of an HIV-fusion inhibitor, a CCR5antagonist, a gp41 ligand, an anti-CD4 antibody, TNX-355, an anti-CCR5antibody, PRO140, BMS-488043, BMS-378806, plerixafor, epigallocatechingallate, viriviroc, maraviroc, aplaviroc, griffithsin, JM 3100, TAK 779,TAK-220,4,4-difluoro-N-((1S)-3-(exo-3-(3-isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-8-azabicyclo(3.2.1)oct-8-yl)-1-phenylpropyl)cyclohexanecarboxamide,873140 compound, CP32M and DCM205.
 9. The method according to claim 8,wherein the HIV-fusion inhibitor is T20.
 10. The method according toclaim 1, wherein the amino acid sequence of the AD moiety is selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ IDNO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ IDNO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ IDNO:82, SEQ ID NO:83, SEQ ID NO:84 and SEQ ID NO:91.
 11. The methodaccording to claim 1, wherein the regulatory subunit is selected fromthe group consisting of RIα, RIβ, RIIα and RIIβ.
 12. The methodaccording to claim 1, wherein the amino acid sequence of the DDD moietyis selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:90 and SEQ ID NO:92.
 13. The method of claim 1, furthercomprising administering to the subject at least one additional anti-HIVtherapeutic agent.
 14. The method of claim 13, wherein the subject istreated with HAART (highly active antiretroviral therapy).
 15. Themethod of claim 13, wherein the at least one additional anti-HIVtherapeutic agent is selected from the group consisting of T20, CP32M,IZN17, C34, T1144, C52L, CCR5 antagonists, maraviroc, vicriviroc,abacavir, amdoxovir, AOP-RANTES, apricitabine, atazanavir, bevirimat,BMS-378806, calanolide A, CCR5, CD4, ceragenin, cobicistat,cyanovirin-N, darunavir, diarylpyrimidines, didanosine, dolutegravir,efavirenz, elvitegravir, elvucitabine, emtricitabine, epigallotachengallate, festinavir, fosamprenavir, foscarnet, griffithsin, globoidnanA, hydroxycarbamide, indinavir, KP-146, lamivudine, lefinavir,lersivirine, lopinavir, miltefosine, MK-2048, nelfinavir, nevirapine,racivir, raltegravir, ritonavir, saquinavir, selicicib, stampidine,stavudine, Tat antagonists, tenofovir, tipranavir, trichosanthin,TRIM5alpha, vivecon, zalcitabine and zidovudine.
 16. The method of claim1, wherein the antibody or fragment thereof is attached to two copies ofthe AD moiety and the complex comprises four copies of theDDD-conjugated therapeutic agent.